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This study was performed to explore the effective management of bleeding associated with radiofrequency ablation RFA of benign thyroid nodulesMethods Thirtyfive patients with benign thyroid nodules who were treated with ultrasoundguided RFA from July to December at the Third Affiliated Hospital of Sun YatsenUniversity were retrospectively reviewed The technique efficacy bleeding and other complications were assessed during the followup periodResults The mean technique efficacy was 06 at month and 06 at months after the procedure One case of an intranodular haematoma and two cases of voicechange month were observed All patients recovered with corresponding treatmentConclusion Although the incidence of haemorrhage is low serious haematomas are lifethreatening Therefore having a comprehensive understanding of the potential complicationsan accurate clinical strategy and adequate technical skills may prevent or help to properly managethese complicationsKeywordsRadiofrequency ablation benign thyroid nodules haemorrhage management haematomaultrasoundDate received January accepted June 1Department of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Guangzhou China2General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsBo Liu General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province China Jie RenDepartment of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province ChinaEmails renjieguangzhou126com liubojake126comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cIntroductionAssociationfor AdultThyroid nodules are extremely commonand the associated morbidity rate rangesfrom to according to highresolution ultrasound US findings12Mostthyroid nodules are benign andrequire no intervention other than clinicalfollowup According to the AmericanThyroidManagementGuidelinesPatients withThyroid Nodulesand DifferentiatedThyroid Cancer thyroidstimulating hormone suppression therapy for benign thyroid nodules BTNs is not recommendedbecause the potential harm outweighs thebenefit3 Radioiodine therapy was historically an effective treatment for thyroid hotnodules and a possible alternative to surgery Howeverthis technique has beenproven to have uncertain efficacy andsome adverse effects such as hypothyroidrecurrence4“ Surgery may beism orconsideredgrowing BTNs withpressurerelated symptoms neck discomfort cosmetic concerns or decreased quality of life3 At present partialtotal thyroidsurgery is considered the gold standardtreatment Surgeryassociated withnumerous complications such as nerveinjury anaesthesiarelated problemslonghospital stays conspicuous scars haemorrhage and lesions ofthe parathyroidglands78 In addition hypothyroidism isinevitable after totalthyroidectomy andrequires lifelong hormone supplementationHenceincreasingly minimally invasivetherapeutic strategies are currently used totreat BTNs In most cases several thermalablation techniques such as laser ablationmicrowave ablation radiofrequency ablation RFA and highintensity focused UShave been shown to be effective in BTNsAmong these thermal ablation techniquesRFA is the most widely applied910forisRFA of thyroid diseases first reported in“ is considered efficacious and safeJournal of International Medical Researchfor treatment of BTNs1415 To date no lifethreatening complications related to RFAhave been reported Howeverseveralcases of haemorrhagerelated to fineneedle aspiration FNA or core needlebiopsy CNB have been reported16“Although a microinvasive procedure suchas FNA can result in massive uncontrolledbleeding resulting in upper airway respiratoryuncontrolledbleeding is a rare but lifethreatening complication of RFA Thus management ofbleeding associated with RFA of BTNs isof vitalimportance This study was performed to explore the effective managementof bleeding associated with RFA of BTNsobstructionsuchMaterials and methodsofThis study was approved by the EthicsCommitteethe Third AffiliatedHospital of Sun Yatsen University andwritten informed consent was obtainedfrom all patients prior to the performanceof USguided FNA or CNB and RFA Therequirement to obtain informed consent forpublication was waived because of the retrospective nature of the studyPatientsAll consecutive patients who underwentRFA of BTNs at our institution from July to December were analysed Thefollowing inclusion criteria were appliedconfirmation of benignancyBethesdaClass II by FNA cytology or CNB complaints of pressure symptoms compressivesymptoms neck discomfort orforeignbody sensation or cosmetic problems a2cm maximum diameter of the indexnodule anxiety about a malignancy unsuitability for surgery or unwillingness toundergo surgery and a normal serum thyrotropin concentration normal completeblood counts and normal blood coagulation test results The exclusion criteria 0cHu et alwere nodules showing malignant featuresie taller than wide spiculated marginmarked hypoechoic appearance or microcalcifications on US imaging19 abnormalthyroid function performance of othertreatments for the thyroid nodules within months before the procedure pregnancyand age of years For the presentstudy only patients with 15 months offollowup after the procedure were included Thirtyfive patients met the inclusioncriteriaPretreatment assessmentBefore the procedure conventional USfindings USguidedFNA findingscontrastenhanced US CEUS findingsand laboratory and clinical results wereevaluated Two radiologists TW and JR with and years of thyroid US experiencerespectively performed the USUSguided FNA and CEUS examinationsusing a Logiq E9 US device GE MedicalSystems Milwaukee WI USA equippedwithtransducerwith a MHzfrequency range “ MHz The USexamination included characterisation ofthe location shape size margins solidcystic proportions echogenicity calcification status and internal vascularity ofeach nodulefrequency ofan ML615centrelinearLaboratory tests included the levels ofthyroidstimulating hormone free triiodothyronine free thyroxin and thyrotropina complete blood cell count and a coagulation test prothrombin time and activatedpartial thromboplastin time The nodulevolume was calculated using the followingvolume¼ length 02 width 02equationdepth 02 In addition all patientsunderwent vocal cord function assessmentsby an experienced laryngologist before theablation procedure Atenrolment allpatients were asked to rate their pressuresymptoms on a 10cm visual analoguescale grade “ cm and the cosmeticgrading score was assessed by the physicianas described in the consensus statement20Procedures and equipmentpreventsignificantAll RFA procedures were performed by oneradiologist JR with years of experienceperforming RFA in an outpatient clinic Weused an RF generator VIVA RF SystemVR STARmed Gyeonggisi South Korea andan internally cooled 18gauge 70mmlength or 10mm activetip electrodeStar RF ElectrodeVR STARmed Localanaesthesia with lidocaine was appliedto the puncture site The hydrodissectiontechnique was used under US guidance glucose and norepinephrine weremixed and injected into the surroundingthyroid capsule which provided a safe distance between the needle tip and adjacentcritical structures During the procedurewe paid special attention to the preservation of surrounding important structurestocomplicationsTherefore two essential techniques werethe transisthmic approach andappliedtechnique2122 Ablationthe movingshotwas suspended when the index nodule wascovered by hyperechoic zones The technique efficacy TE was then evaluated byCEUS at to minutes after RFA untilthedisappearedTechnicalthechange of an entire nodule to a noenhancement zone on realtime CEUSFor nodules with an enhancement zonean additional ablation was performed todestroy the nodule as much as possibleComplications were monitored immediatelyafter the procedure and during the followup period Major and minor complicationsand adverse effects were defined accordingto the criteria established by the Society ofInterventional Radiology2324success was defined ashyperechoiczones 0cJournal of International Medical ResearchFollowup evaluationatandperformedserum thyroidAny specific complaints or concerns wererecorded for month Postproceduralfollowup wasand months after treatment At each followup visit a US examination CEUS examinationhormonemeasurements were performed pressuresymptoms and the cosmetic grading scorewere evaluated and the volume ofthetreated nodule was calculated The TE wascalculated using the following equationTE¼ final nodule volumeinitial nodule volume 02 Statistical analysisstatistical analyses were performedAllusing SPSS software version IBMCorp Armonk NY USA Continuousvariables are expressed as mean 06 standarddeviation Quantitative data for volumeand TE were analysed using a pairedttest A P value of 14 was consideredstatistically significantResultsThe patients™ characteristics are summarised in Table Thirtyfive patients underwent RFA including male and femalepatients mean age years The meanlargest BTN dimension was 06 mmrange “ mm and the mean BTNvolume was 06 mL Twentytwototal complications minor and majorcomplications were observed among thetreated patients None of these complications was lifethreatening and all occurredwithout sequelaeNodule volumeAfter treatment the overall volume of thesignificantly decreased 06nodules 06 mL at mL at month and 06 mL at monthsbaselineTable Patients™ baseline characteristics n¼ Characteristics 06 06 06 06 06 06 06 06 Age at treatment yearsMalefemale ratioBody weight kgBody height cmBody mass index kgm2Symptom score “Cosmetic score “Cosmetic score of Cosmetic score of Cosmetic score of Preablation serum FT4 level pmolLPreablation serum TSH level mIULIndex nodule on ultrasoundRight sideLeft sideLargest dimension mm to to 15Data are presented as mean 06 standard deviation ornumber of patientsFT4 free thyroxin TSH thyroidstimulating hormoneP and the TE was 06 at month and 06 at months P Table Figure shows the shrinkage of the nodules at and months after the procedure comparedwith baseline no hypoechoic blood supplywas observed within the area ofthenodulesBleeding complicationsTwelve patients developed bleeding complicationsincluding a perithyroidal haematoma minor complication in patientsand an intranodular haematoma majorcomplication in patient as shown inTable The haematomas were detectedby US scans which revealed gradualenlargement of a hyperechoic mass in oraround the nodules Figure For thepatient with intranodular haemorrhage 0cHu et alTable Changes in volume before RFA and at each followup visitParameterInitial month laterLargest diameter mm 06 “ 06 “Volume mLTechnique efficacy ”Data are presented as mean 06 standard deviation range 06 “ 06 “ 06 “ months laterP value 06 “ 06 “ 06 “ Figure a c e Ultrasound examination and b d f contrastenhanced ultrasound examination of a39yearold woman treated with radiofrequency ablation a b Ultrasound and contrastenhanced ultrasound revealed a cysticsolid nodule before ablation c d One month after ablation ultrasound showed ahypoechoic nodule with a decreased volume d e Six months after ablation the volume of the nodule haddecreased further and no blood supply was observed within the area of the nodule 0cJournal of International Medical ResearchTable Complications and adverse effects in patients who underwent RFA of thyroid nodulesComplication or adverse effectAdverse effectsFeverPainDizzinessSensation of heatMinorPerithyroidal haematomaVomitingnauseaOedemaswellingVoice change for monthMajorVoice change for monthIntranodular haemorrhageData are presented as n the haematoma was controlled throughtimely use of the ablation needle to coagulate the injured blood vessel and by injecting lyophilising thrombin powder into thehaematoma Figure Most of the perithyroidalseriesrequired only observation with or withoutcompression and disappeared within to weeks after the procedure None of the patientssubscapularhaematomahaematomasdevelopedthisinaOther complications and adverse effectsThe adverse effects of RFA included fevern¼ pain n¼ dizziness n¼ and a sensation of heatn¼ Minor complications includedoedemaswelling n¼ and a voicechange for month n¼ vomitingnausean¼ DiscussionImageguided thermal ablation techniquessuch as laser ablation ethanol ablationmicrowave ablation highintensity focusedUS and particularly RFA have recentlybecome more widely used to treat thyroidthe creation ofnodules Briefly the basic mechanism ofRFA involvesthermaldamage by friction and heat conductionwhich is generated from an oscillatinghighfrequency alternating electric currentproduced by the RFA generator and thentransferred through the electrode tip Theenergy of RFA is powerful and accurate2526 RFA is considered an effectiveand safe treatment for control of BTNsIn most cases the incidence of haemorrhage and other complications is low20However haemorrhage is sometimes lifethreatening because serious haematomasmay compress the upper airways Manyreports have described active bleedingduring FNA of thyroid nodules and RFAof hepatocellular carcinomas14““ andsome reports have described fatalities14ThusimportantcomplicationhaemorrhageanisThree types of haemorrhage may occurperithyroidal subcapsular and intranodular121427“ The mechanism of haemorrhage is thoughtto be related to themechanical or thermal injuries induced bythe RFA electrode tip3031 Thyroid nodulesreportedly have abundant capsular vesselsthat are usually anastomosed with vesselspenetrating into the core32 These numerousvessels are abnormal thinwalled and susceptible to rupture Large thyroid nodulesare another cause of haemorrhage becausemultiple insertions are often required totreat such nodules In additionthepatient cannot coordinate with the physician during the RFA procedure the perithyroidal orintrathyroidal vessels mayeasily be damaged by movement of theneedle tip or production of heat energyifIt is important to manage bleeding associated with RFA of BTNs Based on ourexperience we suggest several steps to preventshouldobtain a thorough medical history of eachpatient before the procedure All risk factorsdrugssuch bleeding Physiciansincludingbleedingfor 0cHu et alFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage and perithyroidal haemorrhage a Ultrasound and contrastenhanced ultrasoundrevealed a hyperechoic mass lesion in the nodule b Ultrasound and contrastenhanced ultrasound revealedperithyroidal haemorrhagedrugsnonsteroidalantiplateletantiinflammatory drugs and anticoagulantsand diseases affecting coagulation shouldbe recorded33 In addition the patient™scoagulation function should be thoroughlyevaluated All patients with clinical coagulation disorders should be excluded Evenwhen coagulation indices are normalinpatients with high risk factors for bleedingsuch as liver cirrhosis endstage renal disease anticoagulant use or hypertension34sufficient preoperative preparation shouldbe emphasised A patient with active bleedingthesein the presentstudy metconditions Although his coagulation indices were normal he had a subclinical coagulation disorder due to endstage liverdisease Fresh frozen plasma or human prothrombin complex should be used inpatients with liver cirrhosis and anticoagulants should be withdrawn in these patientswhich will help to improve coagulation function before the procedure If a possibility ofbleeding exists Reptilase haemocoagulaseatrox forinjection Pentapharm BaselSwitzerland can be used preoperativelyDuring the RFA procedure an effectiveclinical strategy and adequate technical 0cJournal of International Medical ResearchFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage or perithyroidal haemorrhage during ablation a Ultrasound revealed a hyperechoicmass lesion in the nodule b Ultrasound showed an ablation needle inserted into the nodule to coagulatebleeding vessels c After lyophilising thrombin powder was injected into the hematoma ultrasound andcontrastenhanced ultrasound showed disappearance of the hyperechoic mass lesion and microbubbleextravasation d Ultrasound showed a hyperechoic mass lesion around the thyroid and contrastenhancedultrasound showed no microbubble extravasation around the thyroid e After lyophilising thrombinpowder was injected into the haematoma no microbubble extravasation was observedskills are both essential Patient cooperationis the first requirement When the needle tipis in the patient™s body any uncooperativemotion of the patient may lead to injury ofvessels or other structures Most patientscan endure the procedure under local anaesthesia however anxious patients mayrequire general anaesthesia to achieve cooperation If possible smallbore electrodesshould be chosen to decrease the risk ofbleeding35 It is necessary to cauterise thesupplying vasculature of nodules to avoidrecurrence and residue Howeverthepuncture route should be carefully designedto avoid pericapsular vessels and the electrode tip should be closely monitoredActive bleeding during needle puncture isvisible as a rapidly expanding hypoechoicor anechoic signal Locating the haemorrhagic focus is not difficult with CEUSguidance The bleeding pointcan beblocked by RF electrode tip insertion anddirect ablation When the bleeding is toorapid to control with the RF electrode tipby increasing the power drug injection is asuitable alternative Lyophilised thrombin 0cHu et alpowder can be dissolved in normal salineand then injected at the bleeding pointthrough a syringe with US guidance Onereport also described haemorrhage treatedby local injection of hypertonic saline andepinephrine solution in a patient with hepatocarcinoma36 Mildbleeding whichappears as a hypoechoic layer can mostlybe controlled using ice and compression ofthe neck for several minutes after the procedure30 All bleeding can be controlled byconservative methodsthus no surgicalintervention is needed Ecchymosis can befound after the procedure and usually disappears in approximately to weeksPostprocedure CEUS is indispensablefor all patients regardless of whether bleeding occurs CEUS is an objective evaluationtool for active bleeding37 Close clinicalobservation for hours postoperatively isrecommended in our department becausemost bleeding occurs during the firstlobectomy38Observation of the neck can help to detecta haematoma early and may aid in preventing serious adverse effectsthyroidhoursafterConclusionAcute thyroid bleeding is one possible complication of RFA although rare it is potentially lifethreatening Proper selection ofpatients and sufficient preparation areessential During the RFA procedureboth an effective clinical strategy and adequate technical skills are indispensable Thephysician should trace the electrode tipusing realtime US and sufficiently managebleeding Mild bleeding has limited morbidity and can be easily controlled by compression Active bleeding tends to be rarehowever it may be disastrous if the operator is unaware or careless Direct ablationwith the RF electrode tip and drug injectioninto the bleeding focus are effective modalities for active bleeding CEUS and closeobservation are also recommended afterthe procedureto detect abnormalitiesearly RFA is an effective and relativelysafe alternative for selected patients withBTNs if performed by skilled physiciansAuthor contributionsI Conception and design Jie Ren and Bo LiuII Administrative support Jie RenIIIstudy materials or patientsProvision ofKunpeng Hu and Yufan Lian IV Collectionand assembly of dataJinfen Wang andWenchao Li V Data analysis and interpretation Wenchao Li and Zhicheng YaoVIManuscript writing All authors VII Finalapproval of manuscript All authorsData availabilityData regarding the patients™ characteristics usedto support the funding are shown in Table Declaration of conflicting interestThe authors declare that there is no conflict ofinterestFundingthe NaturalThis work was supported by the NationalNatural Science Foundation of China CNNoScienceFoundation of Guangdong ProvinceNo2016A030313200 the Science and TechnologyProject of Guangzhou City No the Hengrui Foundation of Hepatobiliary andPancreaticNoCXPJJH1180000120183331NaturalScience Foundation of Guangdong ProvinceNothe FundamentalResearch Funds for the Central UniversitiesSun Yatsen University No 17ykpy67 andthe Clinical Research Project of Sun Yatsen University No 2017A030313580theCancerResearchORCID iDKunpeng Huorcid00000001 0cReferences Guth S Theune U Aberle J et al Very highprevalence of thyroid nodules detected byhigh frequency MHz ultrasound examination Eur J Clin Invest “ Tan GH and Gharib H Thyroid incidentalomas management approaches to nonpalpable nodules discovered incidentally onthyroid imaging Ann Intern Med “ Haugen BR Alexander EK Bible KC et al AmericanThyroid AssociationManagement Guidelines for Adult Patientswith Thyroid Nodules and DifferentiatedThyroid Cancer The American ThyroidAssociation Guidelines Task Force onThyroid Nodulesand DifferentiatedThyroid Cancer Thyroid “ Ceccarelli C Bencivelli W Vitti P et alOutcome ofradioiodine131 therapy inhyperfunctioning thyroid nodules a years™ retrospective study Clin EndocrinolOxf “ Reiners C and Schneider P Radioiodinetherapy of thyroid autonomy Eur J NuclMed Mol Imaging S471“S478 Nieuwlaat WA Hermus AR SivroPrndeljF et al Pretreatment with recombinanthuman TSH changes the regional distribution of radioiodine on thyroid scintigramsof nodular goiters J Clin Endocrinol Metab “ LinosDEconomopoulosKPKiriakopoulos A et al Scar perceptionsafter thyroid and parathyroid surgery comparison of minimaland conventionalapproaches Surgery “ Jeannon JP Orabi AA Bruch GA et allaryngeal nervethyroidectomy a systematicDiagnosis ofpalsy afterreview Int J Clin Pract “recurrent Lang B Woo YC and Chiu KW Identifyingpredictive factors for efficacy in high intensity focused ultrasound HIFU ablationof benign thyroid nodules “ a retrospectiveInt J Hyperthermia analysis“ Mauri G Pacella CM Papini E et alImageguided thyroid ablation proposalforterminology andstandardization ofJournal of International Medical Researchreporting“criteria Thyroid Sato M Tateishi R Yasunaga H et alMortality and hemorrhagic complicationsassociated with radiofrequency ablation fortreatment of hepatocellular carcinoma inendstagepatients on hemodialysissurveyrenalJ“Gastroenterol Hepatolnationwidediseasefora Krokidis M Spiliopoulos S Jarzabek Met al Percutaneous radiofrequency ablationof small renal tumours in patients with asingle functioning kidney longterm resultsEur Radiol “ Lim HK Lee Dupuy DE Monchik JM Decrea C et alRadiofrequency ablation of regional recurrence from welldifferentiated thyroid malignancy Surgery “JH Ha EJalRadiofrequency ablation of benign nonfunctioning thyroid nodules 4year followup results for patients Eur Radiol “et Braga M Cavalcanti TC Collaco LM et alEfficacy of ultrasoundguided fineneedleaspiration biopsy in the diagnosis of complex thyroid nodules J Clin EndocrinolMetab “ Kakiuchi Y Idota N Nakamura M et alA fatal case of cervical hemorrhage after fineneedle aspiration and core needle biopsy ofthe thyroid gland Am J Forensic Med Pathol “ Donatini G Masoni T Ricci V et al Acuterespiratory distress following fine needleaspiration of thyroid nodule case reportand review of the literature G Chir “ Roh JL Intrathyroid hemorrhage and acuteupper airway obstruction after fine needleaspirationthyroidglandLaryngoscope “theofofAssociation Gharib H Papini E Garber JR et alAmericanClinicalEndocrinologists American College ofEndocrinology and Associazione MediciEndocrinologi medical guidelines for clinicalpractice for the diagnosis and managementthyroid nodules“ update EndocrofPract “ 0cHu et al Na DG LeeetJHJung SLalRadiofrequency ablation of benign thyroidnodules and recurrent thyroid cancers consensusstatement and recommendationsKorean J Radiol “ Ha EJ Baek JH and Lee JH Movingshotversus fixed electrode techniques for radiofrequency ablation comparison in an exvivo bovine liver tissue model Korean JRadiol “ Jeong WK Baek JH Rhim H et alRadiofrequency ablation of benign thyroidnodules safety and imaging followup in“patients Eur Radiol Cardella JF Kundu S Miller DL et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S189“S191 Sacks D McClenny TE Cardella JF et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S199“S202 Goldberg SN Radiofrequency tumor ablation principles and techniques Eur JUltrasound “ Rhim H Goldberg SN Dodd GR et alEssential techniques for successful radiofrequency thermal ablation of malignanthepatic tumors Radiographics Spec No S17“S35 S36S39 Korkusuz Y Erbelding C Kohlhase Ket al Bipolar Radiofrequency Ablation ofBenign Symptomatic Thyroid Nodules initial Experience Rofo “ Garberoglio R Aliberti C Appetecchia Met al Radiofrequency ablation for thyroidnodules which indications The first Italianopinion statement J Ultrasound “ Baek JH LeeJH Sung JYet alComplications encountered in the treatmentof benign thyroid nodules with USguidedradiofrequencya multicenterstudy Radiology “ablation Chen MH Dai Y Yan KalIntraperitoneal hemorrhage duringandafter percutaneous radiofrequency ablationof hepatic tumors reasons and managementChin Med J Engl “et Rhim H Dodd GR Chintapalli KN et alRadiofrequency thermal ablation of abdominal tumors lessons learned from complications Radiographics “ Terry WI Radium emanations in exophthalmic goiter”blood vessels of adenomas ofthyroid J Am Med Assoc “ Hor T and Lahiri SW Bilateral thyroidhematomas after fineneedle aspiration causing acute airway obstruction Thyroid “ Minami Y Hayaishi S and Kudo MRadiofrequency ablation for hepatic malignancies is needle tract cauterization necessary for preventing iatrogenic bleeding DigDis “ Baek JH Kim YS Lee D et al Benign predominantly solid thyroid nodules prospective study of efficacy of sonographicallyguided radiofrequency ablation versus control condition AJR Am J Roentgenol “ Koda M Murawaki Y Hirooka Y et alComplications of radiofrequency ablationfor hepatocellular carcinoma in a multicenter study an analysis of treated nodules in patients Hepatol Res “ Wiggermann P Wohlgemuth WA Heibl Met al Dynamic evaluation and quantificationof microvascularization during degradablestarch microspheres transarterial chemoembolisation DSMTACE of HCC lesionsusingultrasoundCEUS a feasibility study Clin HemorheolMicrocirc “enhancedcontrast Rosenbaum MA Haridas M and McHenryCR Lifethreatening neck hematoma complicating thyroid and parathyroid surgeryAm J Surg “ 0c'

Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earth™s species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleep™s quotidian œnormalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brain™s clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patient™s desired sleep time or augmenting factors that entrainthe body™s clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require “ h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon œsiesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period œCatchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances œmorning larks latephase delays œnight owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of “ weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ‰¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway™s soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisorders”specifically sleep walking”also occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women “ in males and “ in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [“] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economo™s encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while œasleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of “ OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in œthe loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleep”or its encephalopathic analog”may affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i

high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with łukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in œomics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a łukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a œsymptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that œsymptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie łukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see œmembership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in œfuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of thełukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see œfuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see œdendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 ˆˆ[ ] and lettingd be a dissimilarity relation which is obtained from r bydx y ˆ’ rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie łukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute sˆ— if sˆ—ielse ci sti si ˆª si —¦ sicid8 si replace si with sˆ—i sˆ—i and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ‰¤ i ‰¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let aˆ— be the identity matrixb for each ci calculate e aˆ— aˆ— ˆª aˆ— —¦ cic if aˆ— is not changed a aˆ— and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the χ2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the χ2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have χ2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logrank™s test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logrank™s test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ˆ’ ] to determine the number of clusters for a method we used the œelbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ˆ’ ] ˆ’ 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrank™stest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of ˆ’log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean ˆ’log10 of logrank™s testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg œhigh œmedium œlow in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of “log10 of logrank™s testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes a“c results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors™ contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliœparthenope tecniche di machine learning e soft computing per l™elaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli œparthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id “serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics “rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res “reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs “ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci “napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason “ciaramella a cocozza sand clustering of genomic data neural netw “iorio f miele g napolitano f pinelli m raiconi g tagliafer

" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from ˆ’ to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors™ contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didn™t involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am “biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw “simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol “huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem “liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg “xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett “zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med “ binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med“ yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a

" gastric neoplasms containing neuroendocrine carcinoma nec components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure nec and mixed tumors containing neccomponents we aimed to investigate whether there is a distinct difference in overall survival os between gastricneoplasms containing nec components and gastric adenocarcinomamethods surgically resected gastric neoplasms containing nec components n and gastricadenocarcinomas n from january to december at peking university cancer hospital wereretrospectively analysed patients were categorized into a surgical group and a neoadjuvant group and adjustedusing propensity score matching in the two groups gastric neoplasms containing nec components were dividedinto pure nec and mixed tumors with less than ghminen between and ghminen andmore than ghminen neuroendocrine carcinoma components os was compared between thesegroups and the gastric adenocarcinoma groupresults the os of gastric neoplasms containing neuroendocrine nec components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or cox multivariable regressionanalysis suggested that tumor category neoplasms containing nec components or gastric adenocarcinoma wasan independent risk factor for prognosis interestingly among patients receiving neoadjuvant therapy thedifference was not significantcontinued on next page correspondence buzhaodecjcrcn jijiafuhscpkueducn jiahui chen anqiang wang and ke ji contributed equally to this workdepartment of gastrointestinal surgery key laboratory of carcinogenesisand translational research ministry of education peking university cancerhospital institute no fucheng road haidian district beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchen bmc cancer page of continued from previous pages gastric neoplasms containing any proportion of nec components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmskeywords neuroendocrine carcinoma gastric adenocarcinoma overall survival gastric neoplasms containing neuroendocrine carcinomanec components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure necs and mixed tumorscontaining nec components every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing nec components account for approximately “ of thesecases [ ] given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory [“]according to the world health anizationwho digestive neuroendocrine tumor classificationneuroendocrine neoplasm nen can be divided intothree categories based on ki67 levels and mitotic counts— hpf grade g1 ki67 ‰ mitoses grade g2 ki67 ‰ ‰ mitoses‰ grade g3ki67 mitoses meanwhile the americanjoint committee on cancer ajcc defines highly differentiated nen as a neuroendocrine tumor net and thepoorly differentiated nen as a neuroendocrine carcinoma nec based on the degree of tumor cell differentiation generally g1 g2 and rare welldifferentiated g3nens belong to the nets while poorly differentiatedg3 nens belong to necs[ ] gastric mixedneuroendocrinenonneuroendocrineneoplasm gminen is a special type of gastric nen that is definedas containing more than of both neuroendocrineand nonneuroendocrine components accountingfor approximately of all gnens and of gastricneuroendocrine carcinomas gnecs [“] for thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition consideringthe heterogeneity ofminen and the malignancy degree of the different components in the tumor la rosa [ ] proposeddividing minen into three categories highgradeintermediategrade and lowgrade highgrade minenconsists of nec and carcinomaadenoma intermediategrade mimen consists of net and carcinoma and lowgrade minen consists of net and adenoma thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm ghminen was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandgenerally the prognosis of mixed tumors is largely determined by the most malignant component kim found that gnec has shorter progressionfree survival pfs than gastric adenocarcinoma huang found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components all of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone we sought tofind studies on the overall survival os comparison between ghminen and gastric adenocarcinoma butfailed thus we think that a study of the comparison ofthe os of ghminen and gastric adenocarcinoma willprovide a valuable supplement to current research on ghminen to overcome the bias caused by the differences between the covariates in the comparison we usedpropensity score matching psm to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliablemethodspatient selectionwe retrospectively collected patients diagnosed withgastric nens and underwent radical resection at pekinguniversity cancer hospital beijing from january to december the inclusion criteria were as follows pathologically confirmed pure nec or tumorcontaining nec components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup patients diagnosedwith cm1 or ct4b before surgery or died from perioperative complications were excluded from the study 0cchen bmc cancer page of patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for psm analysesperformed the chisquared test and mannwhitney utest were used to further verify the matching resultsfollowupwe followed the patients at least twice a year serumtumor markers test gastroscope and computed tomography ct scans were used to reexamine patients aftersurgery depending on the patients™ status magneticresonance imaging mri and positron emission tomography computed tomography petct were alsoconsidered for patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationdiagnosis and classificationwe reevaluated the diagnosis and classification of ghminen mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as ghminen and ghminenrespectively atumor consisting of nec is defined as pure necall neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the who classification of tumors neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin syn chromogranina cga and neuro cell adhesion molecule cd56 orncam the tumor staging described in the study wasbased on the ajcc 8th edition tnm staging guidelines all possible disagreements were discussed in ourstudy groupdefinition of variables and groupsin this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery patients inthe surgery group were assessed by the ptnm stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the yptnm staging system osrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egpropensity score matchingto accurately compare the prognosis of ghminenand gastric adenocarcinoma we employed psm to balance the differences between the two groups psm wasperformed through the pamatching plugin in spss software logistic regression models were used toestimate propensity scores based on gender age tumorlocation tumor size and pathological staging given a caliper width nearest neighbor matching wasstatistical analysisall statistical analyses were performed using spss statisticalsoftware ibm united states the chisquared test and mannwhitney u test were used forstatistical analysis of categorical variables and continuous variables respectively kaplanmeier method wasused for the comparison of os the logrank test wasused to compare survival rates multivariable cox proportional hazards models were used to identify predictors of survival outcome p was regarded as thethreshold of significanceresultspatient selection and psm resultsbetween and among the patients treated atthe gastrointestinal cancer center of peking universitycancer hospital a total of patients with gastric neoplasms containing nec components met the inclusioncriteria for the study including cases of pure necand cases of mixedtype of these patients a total of patients received neoadjuvant therapy nec ghminen ghminen ghminen while the remaining patients receivedsurgery directly nec ghminen ghminen ghminen there were aninsufficient number of patients in group ghminen group to conduct effective statistical analysisso we combined the ghminen group with thenec group for further analysis we also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly fig immunohistochemical specificity markers were utilizedto identify the neuroendocrine components fig 2asyn was expressed in almost all neoplasms containingnec components while the positive rates ofcga and cd56 were much lower and respectively no significant difference in the positiverate of syn and cga was observed between pure nec ghminen ghminen and ghminenfig 2b c only the positive rate of cd56 was found tobe higher in the pure nec group than that in the ghminen group fig 2dtherefore priorto os comparison psm wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cchen bmc cancer page of fig flow chart of patient enrolmentcomparison of os between all patients with neccomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupbefore psm we compared the survival curves between all patients with nec components and patientswith gastric adenocarcinoma by the kaplanmeiermethod fig apparently patients with nec components had a poorer os than those with gastricadenocarcinoma fig 3a p in the surgicalgroup in contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy fig 3b p according to the proportion of nec components patients were classifiedinto pure nec ghminen ghminenand ghminen the os was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison fig 3c dfig illustrations of immunohistochemical staining patterns in gastric neoplasms containing nec components a an overview of the expressionof syn cga and cd56 in tumors containing nec components b syn expression in different nec component groups c cga expression indifferent nec component groups d cd56 expression in different nec component groups cd56 neuro cell adhesion molecule cgachromogranin a nec neuroendocrine carcinoma syn synaptophysin pvalue 0cchen bmc cancer page of fig see legend on next page 0cchen bmc cancer page of see figure on previous pagefig comparison of os between gastric neoplasms containing nec components and gastric adenocarcinoma a os comparison betweengastric neoplasms containing nec components and gastric adenocarcinoma before psm in the surgical group b os comparison between gastricneoplasms containing nec components and gastric adenocarcinoma before psm in the neoadjuvant group c os comparison between differentnec content groups pure nec ghminen ghminen and ghminen and gastric adenocarcinoma before psm in the surgicalgroup d os comparison between the different nec content groups and gastric adenocarcinoma before psm in the neoadjuvant group e oscomparison for patients in the surgical group after psm f os comparison for patients in the neoadjuvant group after psm nec neuroendocrinecarcinoma os overall survival psm propensity score matchingbefore psm significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group table table to balance the clinicopathological differences between the twogroups psm was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups the detailedclinicopathological characteristics before and after psmare shown in table and table as a result patients with nec components and patients with gastric adenocarcinoma were matchedin the surgical group table patients with nec components also had a poorer os than those with gastricadenocarcinoma fig 3e p multivariable analysis showed that adjuvant therapy tumor category andtnm stage werefactorstable independent prognosticto investigate whether neoadjuvant therapy had an effect on os patients with nec components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group table interestingly kaplanmeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in os between the two groups fig 3f p comparison of os between patients with differentproportions of nec components and patients with gastricadenocarcinomato investigate whether the level of nec componentshad an effect on os in the surgical group ghminen ghminen pure nec and pure nec plus ghminen were compared with gastric adenocarcinoma after psm the results showed that even thegroup with the lowest proportion of nec componentsthe ghminen group had a poorer os thanadenocarcinoma fig 4a p as expected theghminen pure nec and pure nec plus ghminen groups each with relatively high proportionsof nec components had worse os than the gastricadenocarcinoma group fig 4bd p detailed clinical information after matching isshown in additional file tables s1s4psm was also performed in the neoadjuvant group incontrast to the results of the surgery group in the purenec group containing the highest proportion ofnec componentstill no significantdifference in os from gastric adenocarcinoma fig5d the other three groups with lower nec contentwere also notfrom gastricadenocarcinoma in terms of os fig 5ac detailedclinicopathologicaland afterpsm are shown in additional file tables s5s8characteristics beforethere wassignificantly differentdiscussionamong gastric neuroendocrine neoplasms the tumorcontaining nec components is a special type includingpure nec and mixed tumor containing nec components the incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated gnens [ ]received neoadjuvantin previous study kim found that in patientschemotherapywho had notprogressionfree survivalpfs of pure gnec waspoorer than that of gastric adenocarcinoma while thepfs of mixedtype tumors was not significantly differentin kim™sfrom that of gastric adenocarcinoma study the mixed type was defined as net mixed withgastric cancer rather than nec net is much less malignant than nec [ ] this may be the reason whythere was no significant difference in os between mixedtype and gastric adenocarcinomas in addition mixed tumors with less than or more than of nec components were not included in that study which webelieve was a deficit of the study pfs is an important indicator for evaluating prognosis in many cases it can reflect the trend of os based on kim™s research resultswe regarded tumors containing nec components as awhole and found that the os of these tumors was poorerthan that of adenocarcinoma in the surgical group inthe comparison of os between mixed tumors with different proportions of nec components and gastricadenocarcinoma the results for pure nec cases wassimilar to kim™s while the os of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in kim™s study cox multivariable regression analysis showed thattumor categoryneoplasm with nec component or adenocarcinoma 0cchen bmc cancer page of table comparison of clinicopathological characteristics before and after psm in surgical grouppatient characteristicsunmatched comparisonpatients with neccomponents n p valuematched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm‰¥ cmtype of gastrectomytotal gastrectomydistal gastrectomy ± ± proximal gastrectomy surgical procedureopenlaparoscopict staget1t2t3t4n stagen0n1n2n3m stagem0m1ptnm stageiiiiiiiv gastricadenocarcinoman ± ± ± ± p value gastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer table comparison of clinicopathological characteristics before and after psm in neoadjuvant groupmatched comparisonpatient characteristicsunmatched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm‰¥ cmtype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedureopenlaparoscopict staget0t1t2t3t4n stagen0n1n2n3m stagem0m1yptnm stageiiiiiiiv ± ± gastricadenocarcinoman ± ± p valuepatients with neccomponents n ± ± page of p valuegastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer page of table univariate and multivariate analyses of survival after psm in surgical grouppatient characteristicsunivariate analysishr ci“multivariate analysishr cip valueage yeargendermale vs femalebmiadjuvant therapyyes vs notumor size‰¥ cm vs cmtumor categorycarcinoma with nec component vsgastric adenocarcinoma vstype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedurelaparoscopic vs opentnm stageiiiiiiivp value“““ ““““““““““ “ ““““““““tumor size and tnm staging were independent risk factors for prognosis this suggests that the prognosis ofgastric neoplasms with nec components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of nec components can alsoimpair prognosis which challenges the current cutoffvalue of the proportion of each component that must theoretically be greater than was set in andsince who has also adopted this standard to define minen this largely avoids the overdiagnosisof minen in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges in additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines nevertheless it is now being questionedby an increasing number of scholars the componentsin mixed tumors are not evenly distributed for large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely park compared the os between tumors with morethan nec components and gastric adenocarcinomawith or without less than nec and they found thattumors with an nec composition of more than hada worse prognosis this suggests that even a small proportion of malignant components can affect prognosis while in park™s study for unknown reasons the authors did not compare the prognosis of mixed tumorswith nec components less than with gastricadenocarcinomas directly nor did they compare allneccontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyin our study we regarded tumors containing neccomponents as a whole and found that the os of thesetumors was poorer than that of adenocarcinoma in thesurgical group in addition we also found that the os ofmixed tumors with less than between and more than nec components or pure nec wasworse than that of gastric adenocarcinoma analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of syn and cgabetween different nec content groups only the positiverate of cd56 was found to be higher in the pure necgroup than that in the ghminen group therole of cd56 in the diagnosis of nec is still controversial however syn and cga are two wellrecognized 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec and gastric adenocarcinoma in the surgical group aoverall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparison between ghminen andgastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastric adenocarcinoma d overall survivalcomparison between pure nec alone and gastric adenocarcinomamarkers therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing nec componentsstudies on the molecular mechanism of pathogenesisshow that nec components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity loh and mutations at multiple loci and key oncogenes such as tp53 apc and rbgenes all these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[“] moreoverin the who definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid no minimumpercentage for either ingredient is established thereforewe believe that mixed tumors containing nec components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma we propose considering mixed tumorscontaining nec components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiespreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] in a retrospective study involving patientsma found that neoadjuvant chemotherapy improves the survival of patients with nec and hminenof the stomach van der veen reported that 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec components and gastric adenocarcinoma in theneoadjuvant group a overall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparisonbetween ghminen and gastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastricadenocarcinoma d overall survival comparison between pure nec and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing nec components however because only eight patients wereincluded in the neoadjuvant group van™s results arequestionable in our study among patients receivingneoadjuvanttherapy no significant difference in osbetween mixed tumor and gastric adenocarcinoma wasobserved even for the pure nec group with the highestnec contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsalthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research in addition we proposed treatingall gastric neoplasms containing nec components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms in the futurewe will conduct more genomics studies to confirmour ideas this study also has its limitations due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens as a retrospective study despite our performing psm in advance selection biascannot be completely avoided in addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cchen bmc cancer page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with nec componentless than so we could only treat all mixed tumors withnec component as a wholesour study demonstrated that gastric neoplasms withnec components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072817additional file table s1 comparison of clinicopathologicalcharacteristics before and after psm of 30ghminen patients insurgical group table s2 comparison of clinicopathologicalcharacteristics before and after psm of ghminen patients in surgicalgroup table s3 comparison of clinicopathological characteristics beforeand after psm of 70ghminen plus pure nec patients in surgicalgroup table s4 comparison of clinicopathological characteristics beforeand after psm of pure nec patients in surgical group table s5 comparison of clinicopathological characteristics before and after psm of 30ghminen patients in neoadjuvant group table s6 comparison ofclinicopathological characteristics before and after psm of ghminen patients in neoadjuvant group table s7 comparison of clinicopathologicalcharacteristics before and after psm of 70ghminen plus pure necpatients in neoadjuvant group table s8 comparison of clinicopathological characteristics before and after psm of pure nec patients in neoadjuvant groupabbreviationsajcc american joint committee on cancer ct computed tomography ghminen gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm gnec gastric neuroendocrine carcinoma hpf high power fieldminen mixed neuroendocrinenonneuroendocrine neoplasmnec neuroendocrine carcinoma nen neuroendocrine neoplasmnet neuroendocrine tumor mri magnetic resonance imaging os overallsurvival petct positron emission tomography computed tomographypfs progressionfree survival psm propensity score matching who worldhealth anizationacknowledgmentsthanks to dr zhongwu li of the department of pathology peking universitycancer hospital and his colleagues for their assistance in pathologicaldiagnosis and review thanks to all colleagues in the department ofgastrointestinal surgery of peking university cancer hospital and dr jianghong from the statistics department for their assistance in this studyauthors™ contributionsall authors contributed to the study conception and design jc performeddata collection and wrote the manuscript aw wrote and t revised hemanuscript kj helped with statistical analysis and prepared the illustrationszb edited the manuscript jj conceived the study and reviewed themanuscript all authors read and approved the final manuscriptfundingthis work was supported by the national science foundation for youngscientists of china beijing youth talent plan qml20191101 andscience foundation of peking university cancer hospital “ thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics committee of peking universitycancer hospital and the patients™ written consent was also obtained writteninformed consent for publication was obtained and stored in pekinguniversity cancer hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ matsubayashi h takagaki s otsubo t iiri t kobayashi y yokota t advanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy gastric cancer “park jy ryu mh park ys park hj ryoo by kim mg prognosticsignificance of neuroendocrine components in gastric carcinomas eur jcancer “la rosa s inzani f vanoli a klersy c dainese l rindi g histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms hum pathol “ishida m sekine s fukagawa t ohashi m morita s taniguchi h neuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis am j surg pathol“rayhan n sano t qian zr obari ak hirokawa m histological andimmunohistochemical study of composite neuroendocrineexocrinecarcinomas of the stomach j med investig ““jiang sx mikami t umezawa a saegusa m kameya t okayasu i gastriclarge cell neuroendocrine carcinomas a distinct clinicopathologic entityam j surg pathol “ohike nan la rosa s who classification of tumours of endocrineans 4th ed lyon iarc press amin mb edge sb ajcc cancer s

"The second patient with EGFR mutation achieved the longest PFS and OS (727 and 1249 days respectively). .0087629.g007 Kaplan-Meier estimates of PFS and OS. No statistically significant difference (P?=?0.007) in PFS was observed between metabolic non-progressive (mNP) patients (median PFS 292 days ; range 190“727) and metabolic (mP) progressive patients (median PFS 64 days ; range: 37“216). Improved PFS in non-progressive patients was associated with prolonged OS (mNP; n?=?4; median OS: 1031 days ; 296 to 1249 days versus mP; n?=?8 ; 337 5 days ; 71 to 734 days) (HR 0.34; 95% CI 0.06 to 0.84; P?=?0.03). Discussion Despite the widespread use of [18F]FDG-PET/CT in NSCLC staging a large-scale study recently failed to confirm an overall survival gain in NSCLC patients.[17] This result highlights the value of [18F]FDG-PET/CT in unmet clinical needs such as prediction of residual NSCLC after surgery[18] neoadjuvant therapy[19] or antineoplastic therapy.[20] Prediction of response to antineoplastic therapies would appear to be particularly adapted to targeted therapies that do not induce rapid tumor shrinkage. NSCLC preclinical models have validated this hypothesis with both gefitinib[21] and erlotinib.[22] This original method could compensate for the weakness of RECIST criteria and has led to the proposal of evaluation of new criteria by addition metabolic evaluation by FDG-PET to CT scan.[23] The value of PET in evaluation of response to new targeted therapies emerged in the early 2000 s with the first reports on the efficacy of imatinib mesylate in Gastro Intestinal Stromal Tumor (GIST). Subsequently many studies have confirmed that PET is able to identify very early (i.e. only 24 hours after initiation of treatment) a decrease in glucose metabolism which is correlated with overall survival and progression-free survival of patients with GIST.[24] [25] In the present exploratory study a decrease in SUVmax of at least 21.6% soon after starting therapy (9±3 days) was able to discriminate progressive from non-progressive patients and was associated with improved PFS and OS. This result confirms the results of Mileshkin et al. who showed in a series of 51 patients receiving second- or third-line treatment with erlotinib that an early (14 days) [18F]FDG-PET partial metabolic response was associated with improved PFS and OS even in the absence of subsequent RECIST response.[26] Evaluation of response by [18F]FDG-PET can be performed semi-quantitatively for instance by establishing a SUV cut-off to discriminate metabolic progressive patients from non-metabolic progressive patients. This patient classification (mP/mNP) seems to be more appropriate to assess response to cytostatic therapy that is designed to stabilize disease rather than achieve complete response. The main difficulty of this approach is the overlap of SUV changes between mP and mNP patients. Furthermore different cut-off variations can be expected depending on the types of SUV measured the types of drugs used and the types of tumors which increase the difficulty of establishing a reliable SUV cut-off. However despite the absence of consensus on the most appropriate cut-off value it is generally admitted that the rationale for metabolic response or non-progression of tumor is decreased [18F]FDG tumor uptake or at least stability of tumor uptake over time respectively. Another limitation of semi-quantitative analysis of FDG-PET is that it does not take into account the development of new lesions. However PET detection of new lesions early in the course of therapy has been reported to be a strong independent predictive factor of OS in NSCLC patients treated by EGFR inhibitor.[27] Our findings are consistent with this observation as new lesions occurred in 2/8 patients correctly classified as progressive on PET2 and in 4/5 patients correctly classified as progressive on PET3. One patient (patient #7) was reclassified as mP on PET3 due to the appearance of a new lesion despite a decrease of SUVmax to below the cut-off value. As in our study previous studies failed to demonstrate any difference between SUVmax and SUVpeak.[22] [28] However SUVmax remains the standard for semi-quantitative [18F]FDG-PET assessment probably because is a parameter that can be reliably reproduced by independent operators. It is noteworthy that in our study no significant difference in mean SUV values was observed between PET1 PET2 and PET3 which can be explained by the nature of the cytostatic therapy. 11/12 patients were correctly classified (P versus NP) by PET2 and 10/10 were correctly classified by PET3 by applying the SUV cut-off determined by ROC analysis. In 9/10 patients PET3 revealed response information concordant with PET2. The only patient with discordant [18F]FDG-PET findings was classified by SUV analysis as progressive on PET2 and non-progressive on PET3. Blood glucose injected dose or uptake time were normal and/or not significantly different between PET2 and PET3 (1.16 and 1.4 g/l; 261 and 262 MBq; 60 and 75 min respectively) excluding any to methodology-related error. A flare-up phenomenon could be proposed as described on several occasions on [18F]FDG-PET during cytotoxic treatments for squamous cell carcinoma in prostate cancer patients with bone metastases[29]“[33] and particularly NSCLC patients treated with erlotinib presenting an osteoblastic bone flare-up response mimicking disease progression.[34] Benz et al also described a case of flare-up on early PET in a NSCLC patient treated by erlotinib.[27] Another explanation is that the P/NP classification probably increases mismatches of response assessments related to a discordant outcome of patients with stable disease.[27] Our results suggest that therapeutic efficacy PFS and OS of erlotinib therapy can be predicted 2 weeks after starting erlotinib. These data are consistent with the data of a retrospective study recently published by Kobe et al.[26] [35] At the present time anticancer therapy is currently monitored in the context of hormone-sensitive cancers by regular assay of tumor markers (such as prostate-specific antigen in prostate cancer). The efficacy of hormonal therapy is reflected by a decrease in blood levels of the marker. When the marker remains elevated hormonal therapy is considered to be ineffective and is therefore stopped. Repeated PET imaging can be considered to be a promising approach to evaluate cancer therapy such as targeted therapies that do not induce tumor shrinkage. This new approach appears to be supported by the results of recent clinical trials. The ˜Tarceva Versus Docetaxel or Pemetrexed for Second Line Chemotherapy of Advanced Stage NSCLC™ (TITAN) trial failed to demonstrate an improvement in OS with erlotinib compared to chemotherapy in unselected NSCLC patients receiving second-line treatment (HR?=?0.96; 95% CI 0.78“1.19; p?=?0.73).[36] In a similar group of NSCLC patients the results of the TAILOR trial indicated a highly significant increase of PFS in favor of docetaxel (HR?=?0.71; 95% CI 0.53“0.95; p?=?0.02) versus erlotinib.[37] We consider that evaluation of the metabolic response to erlotinib could provide useful information to rapidly identify patients in whom erlotinib therapy is ineffective especially in EGFR patients without EGFR-activating mutations or unknown status. [18F]FDG-PET could also become a theranostic tool for clinicians. By stopping ineffective therapy earlier physicians can rapidly propose other drugs to a larger proportion of patients with better performance status. This approach could increase the number of patients included in early trials and accelerate drug development. However no medico-economic study has been conducted to determine whether the additional costs induced by [18F]FDG-PET are compensated by the decreased costs of drug (erlotinib) and medical care induced by side effects. Our study highlights the need for more prospective and randomized studies to evaluate the theranostic use of [18F]FDG-PET for management of erlotinib therapy in NSCLC including medico-economic considerations. Conclusion [18F]FDG-PET performed within two weeks of starting erlotinib therapy (9±3 days) appears to be able to predict morphologic response at 2 months according to RECIST criteria. [18]FDG-PET may be clinically useful for early evaluation of targeted therapies as a theranostic tool. Nathalie BAIZE MD Université d'Angers CHU Angers Pôle des Spécialités Médicales et Chirurgicales Intégrées Département de Pneumologie Angers France References 1 FerlayJ ParkinDM Steliarova-FoucherE (2010) Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer46: 765“78120116997 2 JemalA BrayF CenterMM FerlayJ WardE et al (2011) Global cancer statistics. CA Cancer J Clin61: 69“9021296855 3 Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer Collaborative Group. BMJ311: 899“909 4 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 92“9811784875 5 LynchTJ BellDW SordellaR GurubhagavatulaS OkimotoRA et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. 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J Nucl Med52: 1871“187722065872 29 BjurbergM HenrikssonE BrunE EkbladL OhlssonT et al (2009) Early changes in 2-deoxy-2-[18F]fluoro-D-glucose metabolism in squamous-cell carcinoma during chemotherapy in vivo and in vitro. Cancer Biother Radiopharm24: 327“33219538055 30 MessiouC CookG ReidAH AttardG DearnaleyD et al (2011) The CT flare response of metastatic bone disease in prostate cancer. Acta Radiol52: 557“56121498309 31 KrupitskayaY EslamyHK NguyenDD KumarA WakeleeHA (2009) Osteoblastic bone flare on F18-FDG PET in non-small cell lung cancer (NSCLC) patients receiving bevacizumab in addition to standard chemotherapy. J Thorac Oncol4: 429“43119247091 32 BiersackHJ BenderH PalmedoH (2004) FDG-PET in monitoring therapy of breast cancer. Eur J Nucl Med Mol Imaging31 Suppl 1S112“11715112111 33 MortimerJE DehdashtiF SiegelBA TrinkausK KatzenellenbogenJA et al (2001) Metabolic flare: indicator of hormone responsiveness in advanced breast cancer. J Clin Oncol19: 2797“280311387350 34 LindJS PostmusPE SmitEF (2010) Osteoblastic bone lesions developing during treatment with erlotinib indicate major response in patients with non-small cell lung cancer: a brief report. J Thorac Oncol5: 554“55720357621 35 KobeC SchefflerM HolsteinA ZanderT NogovaL et al (2012) Predictive value of early and late residual 18F-fluorodeoxyglucose and 18F-fluorothymidine uptake using different SUV measurements in patients with non-small-cell lung cancer treated with erlotinib. Eur J Nucl Med Mol Imaging39: 1117“112722526960 36 CiuleanuT StelmakhL CicenasS MiliauskasS GrigorescuAC et al (2012) Efficacy and safety of erlotinib versus chemotherapy in second-line treatment of patients with advanced non-small-cell lung cancer with poor prognosis (TITAN): a randomised multicentre open-label phase 3 study. Lancet Oncol13: 300“30822277837 37 GarassinoMC MartelliO BrogginiM FarinaG VeroneseS et al (2013) Erlotinib versus docetaxel as second-line treatment of patients with advanced non-small-cell lung cancer and wild-type EGFR tumours (TAILOR): a randomised controlled trial. Lancet Oncol14: 981“98823883922 Nucleic Acids Res Nucleic Acids Res nar nar Nucleic Acids Research 0305-1048 1362-4962 Oxford University Press 24970867 4117748 10.1093/nar/gku489 15 Methods Online Integrated RNA and DNA sequencing improves mutation detection in low purity tumors Wilkerson Matthew D. 1 2 * Cabanski Christopher R. 1 3 Sun Wei 2 4 Hoadley Katherine A. 1 2 Walter Vonn 1 Mose Lisle E. 1 Troester Melissa A. 1 5 Hammerman Peter S. 6 7 Parker Joel S. 1 2 Perou Charles M. 1 2 Hayes D. Neil 1 8 * 1Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 2Department of Genetics University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 3The Genome Institute at Washington University St. Louis MO 63108 USA 4Department of Biostatistics University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 5Department of Epidemiology University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 6Department of Medical Oncology Dana-Farber Cancer Institute Boston MA 02215 USA 7Broad Institute of Harvard and MIT Cambridge MA 02142 USA 8Department of Internal Medicine Division of Medical Oncology Multidisciplinary Thoracic Oncology Program University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA *To whom correspondence should be addressed. Tel: +1 919 966 3098; Fax: +1 919 966 1587; Email: mwilkers@med.unc.edu Correspondence may also be addressed to D. Neil Hayes. Tel: +1 919 966 3786; Fax: +1 919 966 1587; Email: hayes@med.unc.edu 01 9 2014 26 6 2014 26 6 2014 42 13 e107 e107 15 5 2014 22 4 2014 14 10 2013 © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited. Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method called UNCeqR that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA ERBB2 and FGFR2). In summary integrating RNA-seq with DNA-WES increases mutation detection performance especially for low purity tumors. cover-date 2014 INTRODUCTION Somatically acquired sequence mutations (nucleotide substitutions insertions and deletions) fuel the initiation and progression of cancer (1). Knowledge of mutations in patient specimens informs therapeutic management (23) and in large patient cohorts provides the basis to assess recurrently altered genes that may drive molecular pathogenesis (14“5). DNA whole exome sequencing (DNA-WES) is currently the popular technology to sequence cancer genomes and has led to an abundance of discoveries in many cancer types (46“8). However detecting somatic mutations by DNA-WES with high sensitivity and specificity remains a challenge (79“10) as evidenced by validation rates of 73% in repeated sequencing and by large inter-rater disagreement among different groups analyzing the same sequencing data (710). The biggest challenge is high quality mutation detection in low purity tumors "

" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are “ nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ “] studies haveshown that exosome mirna signaling promotes tumor the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ “] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as œbait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atcc™s instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at —g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturer™s protocol pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatography“mass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm — mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads„¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to “ bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sds“page with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package student™s ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using œthe database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments™ conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p student™s ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p student™s ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p student™s ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [“] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2“ a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn

" the development of a safe effective reversible nonhormonal contraceptive method for men hasbeen an ongoing effort for the past few decades however despite significant progress on elucidating the functionof key proteins involved in reproduction understanding male reproductive physiology is limited by incompleteinformation on the genes expressed in reproductive tissues and no contraceptive targets have so far reachedclinical trials to advance product development further identification of novel reproductive tractspecific genesleading to potentially druggable protein targets is imperativeresults in this study we expand on previous single tissue single species studies by integrating analysis of publiclyavailable human and mouse rnaseq datasets whose initial published purpose was not focused on identifyingmale reproductive tractspecific targets we also incorporate analysis of additional newly acquired human andmouse testis and epididymis samples to increase the number of targets identified we detected a combined totalof genes for which no previous evidence of male reproductive tractspecific expression was annotated manyof which are potentially druggable targets through rtpcr we confirmed the reproductive tractspecific expressionof novel orthologous human and mouse genes without a reported mouse model of these we ablated fourepididymisspecific genes spint3 spint4 spint5 and ces5a and two testisspecific genes pp2d1 and saxo1 inindividual or double knockout mice generated through the crisprcas9 system our results validate a functionalrequirement for spint45 and ces5a in male mouse fertility while demonstrating that spint3 pp2d1 and saxo1 areeach individually dispensable for male mouse fertilitys our work provides a plethora of novel testis and epididymisspecific genes and elucidates thefunctional requirement of several of these genes which is essential towards understanding the etiology of maleinfertility and the development of male contraceptiveskeywords contraception drug target male reproductive tract paralog sperm maturation spermatidspermatozoa correspondence coarfabcmedu thomasgarciabcmedu1dan l duncan comprehensive cancer center baylor college of medicine baylor plaza houston tx usa3department of pathology and immunology baylor college of medicine baylor plaza houston tx usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crobertson bmc biology page of the world human population reached nearly eight billion people in august this number continues torise and is predicted to reach nearly ten billion by theyear the increasing need to promote familyplanning through the development of reliable contraceptive options available to both men and women is widelyrecognized currently there are numerous contraceptiveoptions available to women however identification of asafe nonhormonal contraceptive option for men is stillan ongoing challenge although several different fertilitycontrol alternatives for men have been investigatednone are currently clinically approved for use our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as theidentification and elucidation of the function of key reproductive proteins is still an ongoing effort identifyingdruggable protein targets expressed in the male reproductive tract has been the focus of numerous studiesdedicated to the development of male contraceptionessentialroletheitsthe mammalian epididymis is a segmented ancomprised of a single highly coiled tubule with functionally and morphologically distinct regions that can besubdivided most simplistically into a proximal centraland distal region conventionally named the caput corpus and cauda regions respectively as mammalianspermatozoa transit through the epididymis they acquire the ability to recognize and fertilize an egg properties that they did not possess upon exiting the testis epididymis”inconsideringaddition to maturing germ cells of the testis and spermatozoa”is a prime target for the development of a malecontraceptive to advance progress towards the development of a nonhormonal male contraceptive several previous highthroughput studies have been published thatidentified a number of human mouse and rat genes astestisspecific or epididymisspecific [ “] in schultz conducted the first study to identify malereproductive tractspecific genes using microarraysthroughgeneexpression analysis of meiotic and postmeiotic spermatogenic cells together with parallel analysis of available data from the ncbi unigene database the authorsidentified mouse genes as testisspecific which included genes with both known and unknown function atthe time in the following years through two additional microarraybased studies of rat testes and purifiedrat testicular cells johnston identified and additional or overlapping genes astestisspecific in as part of the continued effort to identify novel contraceptive targets the newer rnaseqbased transcriptomics methodology was utilized identifying human genes as testisspecific together withantibodybased protein profiling many of these genesaffymetrixbasedgenomewidewere characterized in terms of the spermatogenic cellpopulations showing expression the first highthroughput transcriptomics study to identify epididymisspecific genes was a mouse epididymal transcriptome studyin which rna isolated fromeach of the epididymal segments was analyzed bymicroarray analysisidentifying epididymisspecificgenes with distinct patterns of segmental gene regulation later in additional transcriptome profiling utilizing whole genome microarrays resulted in identification of previously unreported epididymisspecific transcripts inthe mouse and epididymisspecific transcripts inthe rat a significant number of the identified mouseand rat genes in these studies were not known at the timeand only the probe identification numbers were presentedwhen evaluating potential druggability in a targetbased drug discovery process one must consider theprotein properties that are required for safe and effectiveinhibition among the most significant is tissue expression specificity to minimize potential adverse effectsprotein function and whether protein activity or interaction with other proteins is potentially druggable sequence similarity to closely related paralogs that may beubiquitously expressed and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest several noteworthyreview publications have mentioned numerous geneswhose critical functions high expression and specificityto the testes or epididymides make them viable nonhormonal male contraceptive targets [“] howeveramong the identified genes a significant number either are required for fertility but are expressed in nonreproductive tissues or are reproductive tractspecific but when disrupted lead to subfertility ineither case both are ineffective and highly undesirableoutcomes for a potential male contraceptive targettherefore the identification of additional novel male reproductive tractspecific genes would allow for furtheradvances to be made in the quest to develop an effectiveand safe nonhormonal male contraceptivein this study newly acquired and previously published human and mouse rnaseq datasets [ “]were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tractenriched transcripts additional databases obtained from illuminating the druggable genome mouse genome informatics andensembl biomart were utilized to stratify the resultsinto subgroups based on protein druggability and on theavailability of a mouse model numerous reproductivetractspecific and reproductive tractenriched potentiallydruggable targets for which no published mouse modelexists congruent in expression across both mouse and human datasets were identified through our analysis and 0crobertson bmc biology page of verified through conventional polymerase chain reactionpcr we present the data in a manner that should bemost relevant and of substantialinterest to the malecontraceptive development field since identification ofnew targets worthy of consideration for further functionalanalysis in a knockout animal model and potential drugtargeting continues to be of vast importanceresults wethrough ouridentified four novelepididymisspecific genes spint3 spint4 spint5 andces5a and two novel testisspecific genes pp2d1 andsaxo1 worthy offunctional validation in an animalmodel through the crisprcas9 system we generatedfour individual gene knockouts spint3 ces5a pp2d1and saxo1 and one double knockout mouse modelspint45 revealing an essential requirement for spint4and spint5 in male mouse fertility and the potential utility of pursuing spint4 in humans as a nonhormonalcontraceptive targetresultsstudy approaches and datadespite significant advances in our understanding of thehuman and rodent testis and epididymis transcriptomemostly through microarraybased studies no prior studies have utilized purified human testis cells for the identification of human testisspecific transcripts no priorstudies have utilized the more stateoftheart rnaseqbased transcriptomics methodology for analysis of human epididymisspecific transcripts and no prior studieshave utilized rnaseq analysis of rodent reproductivetissues or cells to identify rodent reproductive tractspecific transcripts to address these gaps in knowledgeand to increase the number of identified reproductivetractspecific genes in both species using the most relevant highthroughput transcriptomics methodology weanalyzed in parallel on a custom bioinformatics pipelinea large number of published and newly acquired humanand mouse rnaseq datasets one hundred and sixtytwo previously published human and previously published mouse rnaseq datasets were retrieved from thesequence read archive sra the sra value for eachsample is listed in additional file table s1 and additional file table s2 we also generated new humanand new mouse reproductive tissue rnaseq samplesgeo accession gse150854 the final dataset is comprised of new and previously published human testisdatasets previously published purified humangerm cell datasets [ ] previously published purified human sertoli cell datasets [ ] new and previously published human epididymis segment datasets previously published mouse testis datasets new mouse epididymis datasets previouslypublished purified mouse germ cell datasets [ ]and previously published purified mouse sertoli celldatasets an additional previously publisheddatasets contributed to the nonreproductive humantissues and previously published datasets contributed to the nonreproductive mouse tissues figure 1a b summarizes all the samples acquired for thestudywe performed a principal component analysis tovisualize the variation in the samples after correcting forbatch effects human reproductive and nonreproductivetissues grouped according to sample type the reproductive tissue samples clustered by tissue type whetheror not they were newly generated or acquired from thesra fig 1c mouse data showed a similar variation inthe samples based on the tissue type fig 1d for bothhuman and mouse reproductive tissues samples separated by whether or not the rnaseq was performed onisolated cells or the whole tissue epididymal tissue wasdistinct from testis tissue in both human and mousefig 1c didentified in the mouseto identify potential male reproductive tractspecificdrug candidates we analyzed the aggregated rnaseqdata to find genes that were statistically significant in expression when compared to the nonreproductive tissuethat had the maximum expression for that gene thisgene list was then further refined by filtering for genesthat were lowly expressed in the nonreproductive tissuethat had the maximum expression for that gene tpmless than or equal to for human tpm less than orequal to for mouse finally this tpm filtered listwas then filtered for the genes that had a reproductivetissue or cell expression value greater than or equal to tpm for human or tpm for mouse fig 2aacross all the reproductive tissues candidate geneswere identified in the human and candidate geneswerefig 2badditional file fig s1 additional file table s3 andadditional file table s4 summarize the differentialfold change identity of the nonreproductive tissue withmaximal gene expression based on the differential geneanalysis fdr average and standard deviation tpm expression values and log2 cpm gene expression value forthe human and mouse samples respectively the resultsfrom the fdr and tpm expression value filtering forthe human and mouse samples are summarized inadditional file table s5 and additional file tables6file table s5 andadditional file table s6 report the log2 fold changefor the reproductive tissue or cell of interest comparedto the tissue with maximal gene expression the genesidentifiedandadditional file table s6 pass the filters in at least oneofinadditional file table s5 and additional file tables6 a value of zero for a given gene and fold expressionthe reproductive tissues or cells ofrespectively additionalinterestsamplesin additionalfile tables5 0crobertson bmc biology page of fig see legend on next page 0crobertson bmc biology page of see figure on previous pagefig summary of the human and mouse rnaseq samples used in the identification of novel male reproductive tractspecific drug targets thernaseq samples used in the human a and mouse b analyses are schematically shown principal component analysis was performed on thehuman c and mouse d nonreproductive and reproductive samples separately the colors of the circles next to the tissues listed in a and bcorrespond to the colors used in the circles for the pca in c and d sample size n values in red andor black denote the number of new redand previously published black samples included in our analysiscomparison indicates that for that comparison the genedid not pass the filters the majority of genes weredownregulated in the reproductive tissue ofinterestcompared to the maximal geneexpressing nonreproductive tissue additional file fig s2 from theanalysis the majority of the candidate genes that passedthe fdr and tpm filters were identified in the testis orspermrelated cells in both human and mouse samplesadditional file fig s2the majority of candidate genes identified in ourscreen that were testisspecific were already identified bythe human protein atlas andor our reanalysis offig identification of candidate drug male reproductive gene targets a diagrammatic representation of overall methodology used to identifyreproductive tractspecific candidate genes in humans genes and in mice genes the maximum gene expression was determinedacross all the nonreproductive tissue samples for each gene for a reproductive tissue or cell sample of interest genes were then filtered forsignificance using a false discovery rate fdr of less than or equal to based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest genes that passed the fdr filter werefiltered such that the average tpm expression value of the maximum expressing nonreproductive tissue was less than or equal to tpm andthe average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to tpm b diagrammaticrepresentation of the number of human and mouse candidate genes in terms of the number of orthologs in the opposite species thenumber of genes previously or not previously identified in a prior transcriptomicsbased drug target report the availability and phenotypicoutcome of any reported mouse models and the number of novel genes without a reported mouse model congruent across both speciesthe main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in the numbers inparentheses comprise the total number of candidate genes that are either epididymisspecific or specific to testis and epididymis but not testisandor testis cellspecific only 0crobertson bmc biology page of the hpa testis datasets additional file fig s3 andadditional file table s7 thirtysix out of the genes that were identified across all the human epididymis tissue were also identified by the human combinednewly acquired and previously published datasets testiscandidate gene list finally the majority of the candidategenes identified from the combined newly generated and previously published human testis datasetswere shared with genes identified from the various testiscell datasets we identified more candidate genes in thenewly generated human epididymis tissues compared topreviously published data out of genes wereunique to the newly generated caput samples comparedto only out of genes which was unique to the previously published samples out of genes were uniquein the newly generated corpus samples compared to out of genes which were unique to the previously published corpus samples and genes were unique to thenewly generated cauda samples compared to genes inthe previously published cauda data with no overlap between the two cauda gene lists additional file fig s3and additional file table s7 there were candidategenes that overlapped between the newly generated human testis samples and mouse testis sample gene listswhile there were candidate genes that overlapped between the previously published human testis sample andmouse testis sample gene lists additional file fig s3and additional file table s7 across all human epididymis tissue samplesincluding the newly generated andpreviously published samples there were genes in common with the combined list of candidate genes across allthe mouse epididymis tissue samples there was a smalloverlap between the human and mouse samples when thenewly generated human caput corpus and cauda tissueswere individually compared to the mouse caput corpusand cauda tissues there was an overlap of and forrespectivelyadditional file fig s3 and additional file table s7this trend was continued for the candidate gene lists derived from the previously published human caput corpusand cauda samples when compared to the candidate genelist from the mouse caput corpus and cauda with and genes in common for the caput corpus and caudacomparisons respectively additional file fig s3 andadditional file table s7 additional file table s7 details the genes that are unique and in common for each ofthe comparisonscorpuscaputtheandcaudato assess the potential usefulness of the candidategenes identified in each human reproductive tissue asdrug targets we assigned the genes to a protein familyie gpcr or ion channel the majority of identifiedgenes were not from a traditional drug target family likekinases or enzymes the testis and germ cell datasetsprovided the most potential targets while the epididymisdatasets provided the fewest additionalfile figs4a the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file table s8 the majority of thecandidate genes do not have a reported mouse modeladditional file fig s4b additional file table s9summarizes mouse model availability for each candidategene identified from human reproductive tissues or cellsfigure shows the complete list of novel human geneswithout a reported mouse model as identified in each ofthe respective cell andor tissue datasets digital pcrsheatmap and conventional pcrs demonstrating expression of a subset of the novel human reproductivetractspecific genes without a reported mouse modelthat we identified are shown in figs and respectively additional file fig s5 shows the complete listof previously identified human genes that remain without a reported mouse model as identified in each of therespective cell andor tissue datasets additional file fig s6 shows the complete list of male reproductivetractspecific human genes for which a previously generated mouse model shows male infertility phenotype asidentified in each of the respective cell andor tissuedatasetsreproductive tractspecific genes identified throughhuman datasetsthrough our bioinformatics analysis of previously published and newly acquired rnaseq datasets we identified a total of genes as reproductive tractspecific inhumans fig of these genes genes do not have amouse gene ortholog while genes have a mousegene ortholog fig of those with a mouse geneortholog have a single gene ortholog have thesame symbolin mouse while have a differentsymbol in mouse while have two or more orthologous mouse genes seventysix human genes had “orthologous mouse symbols genes had “ orthologous mouse symbols and genes fam205a krtap106 magea10 or2ag1 pramef11 pramef2ssx2 ssx3 and ssx4b had greater than orthologous mouse symbols “ symbols additional file table s5 of the human genes that we identified asmale reproductive tractspecific have not been previously identified in a transcriptomicsbased male reproductive tractspecific study [ “] the sum of ourhuman data confirms the findings of out of genes from djureinovic after reidentificationof gene symbols from reported affymetrix ids and consideration of orthologous genes mouse to human andrat to human our human data confirm the findings of out of genes from johnston out of genes from schultz out of genes fromjohnston out of genes from johnston 0crobertson bmc biology page of fig two hundred and thirtythree novel human reproductive tractspecific genes that each have mouse orthologous genes but with noreported knockout mouse models the listed genes were identified in one or more datasets as indicated in the venn diagram underlined geneswere also identified in our studies as reproductive tractspecific in mouse genes genes written in blue encode either enzymes kinasesgpcrs ogpcrs transporters transcription factors or proteins involved in epigenetic regulation genes genes written in dark red wereidentified in both testis testis andor testis cell and epididymis genes out of genes from johnston and out of genes from jelinsky of the genes that have a mouse gene ortholog have notbeen previously identified as male reproductive tractspecific and of these human genes currently lackmouse phenotype information based on data obtainedfrom ensembl biomart mgi impc and ncbihuman testisspecificthree hundred and eightysix genes were identified astestisspecific through either the reanalysis of djureinovic testis datasets genes identified analysis ofour de novo testis datasets genes identified orboth additional file table s5 three hundred andthirteen genes were congruent across both datasetswhile genes were uniquely identified through our reanalysis of djureinovic ™s datasets and only genes[ac1363524 ankrd20a1 ankrd62fam230aggtlc2iqcm potec prnt and utf1] wereuniquely identified through our de novo datasetsadditional file table s5 interestingly of the genes we identified through djureinovic ™s reanalyzed datasets were not previously identified in theirreport or any of the other previous reports [ “]of these genes we randomly verified of thesegenes as testisspecific in humans through conventionalpcr fig we also verified through rtpcr an additional genes”such as allc cdkl3 cox7b2or2h1 and sppl2c”that had been identified throughprevious studies additional file fig s7 of the genes identified through either testis datasets havenot been previously identified of these genes haveone or more mouse orthologs and of these genes arelacking reported phenotype information of the novelgenes lacking a reported mouse model genes encodeenzymes adam20 cpa5 dusp21 naa11 plscr2prss38 and triml1 encode transcription factorsbhmg1znf560znf729 encode transporters slc22a14 slc25a52and encode proteins of unknown drug target typetgif2lyfoxr2prdm9 0crobertson bmc biology page of fig representative novel reproductive tractspecific human a and mouse b genes without a reported mouse model the listed genes wereidentified through our studies as reproductive tractspecific in both humans and mice the digital pcr heatmap depicts the average transcriptsper million tpm value per tissue per gene from the indicated human and mouse rnaseq datasets as processed in parallel through ourbioinformatics pipeline the data was obtained from published and newly acquired datasets white tpm black ‰¥ tpm the expressionprofile of the human and mouse housekeeping genes gapdh and eif3l is included as reference for data obtained from published datasetssuperscript values succeeding the sample names reference the publications as follows djureinovic fagerberg guo zhu kumar browne consortium helsel da cruz and zimmermann such as etdb smim36 bend2 btg4 cnbd1dppa2 efcab5 erich6 fthl17 iqcm mroh2bms4a5 oosp2 pnma6e ppp4r3c rbmxl3 rtl9spdye4 spem2 all of these genes are listed in fig and many of these genes are listed in figs andor to the best of our knowledge no prior studies haveutilized purified human testis cells for the identificationof human testisspecific transcripts through our analysis we identified genes as human testisspecificthrough one or more of the human germ cell datasetsbut not through either of the human testis datasetsadditionalfile table s5 seventysix genes wereidentified exclusively through one or more of the fivehuman spermatogonia datasets genes such as anp32dc13orf42 dscr4 or13g1 or2d2 or52e4 ssx2tle7 while genes were identified exclusivelythrough the human spermatocyte datasets genes suchas h2bfm mageb17 mageb18 or2b6 tcp10 andznf709 and genes were identified exclusivelythrough the human spermatid datasets genes suchas ac0132691 clec20a or7e24 pramef2 spata31a3 tmem191c znf679 thirtyfour geneswere identified through all three cell types™ datasetsgenes such as ccdc166 eloa2 fam47a heatr9 and spata31a1 many of these genes are listedin figs andor of the genes identified as human testisspecificthrough one or more of the human germ cell datasets genes have not been previously identified ofwhich have one or more equivalent mouse orthologswith of these genes having not been knocked out inmouse of these novel genes with no mouse model encode enzymes glt6d1 prss48 satl1 sult6b1tmprss7 tpte2 triml2 and ttll8 encodes anepigenetic protein taf1l encode gpcrs gpr156tas2r13 tas2r30 tas2r46 tas2r50 vn1r2 encodes a kinase cdkl4 encode ogpcrs such asor2d3 or3a2 or52e5 or8g5 or10j1and 0crobertson bmc biology page of adrenal glandadiposecolonbrainskeletal musclesmooth musclesmall intestinesalivary glandleukocytespancreasstomachprostatethyroidkidneyspleentestisheartlungliverskincorpusuteruscaudaovarycaputaac0221675ac1876531adam20al6720431bhmg1bsph1bx2760929c1orf105c2orf92c4orf51c16orf95ccdc196ces5acpa5dcaf8l1efcab5erich6etdbfer1l5glt6d1iqca1llcn6magea11mageb6mroh2bnaa11pp2d1ppp4r3cprr23aprr23bprr23cprss38saxo1slc22a14spag11aspag11bspata31a1spata31d1spata31d3spata31d4spint3spint4tcp10l2tex13dtex44tex48tex51tle7tpte2triml1trpc5oswfdc8wfdc9wfdc10awfdc10bwfdc13gapdhskeletal musclesmooth musclesmall intestinestomachprostatethyroidspleentestislungskinkidneyheartlivercorpusuteruscaudaovarycaputadiposebladderbraincoloneyebgm5767gm5294adam201700042g07rikgm4969bsph14933412e24rik4930558k02rik4933424g06rik1700011l22rik1700018b08rikgm6657ces5acpa5pet2efcab5erich6etdfer1l5glt6d14931409k22riklcn6magea4mageb3mroh2bnaa11pp2d14932429p05rikprr23a3prr23a3prr23a3prss38saxo1slc22a14spag11bspag11bspata31spata31d1bspata31d1bspata31d1bspint3spint4spint5tcp10ctex13c1tex44tex48gm35060tle7tptetriml1trpc5oswfdc8wfdc9wfdc10wfdc10wfdc13hprtfig rtpcr confirmation of reproductive tract specificity in both humans a and mice b the genes listed in this figure are novel as identifiedthrough our studies and without a reported mouse model humans do not have an equivalent proteincoding equivalent to mouse spint5 gapdh and hprt are included as housekeeping genes 0crobertson bmc biology page of fig three hundred and two novel mouse genes with human orthologs without a reported mouse model the listed genes were identified inone or more mouse datasets as indicated in the venn diagram underlined genes were also identified through our studies as reproductive tractspecific in human genes genes written in blue encode either enzymes kinases gpcrs ogpcrs transporters transcription factors orproteins involved in epigenetic regulation genes genes written in dark red were identified in both testis testis andor testis cell andepididymis genesnkx24or14a2 encode transcription factorsznf99 znf648 znf705b znf705g and znf709 encode transporters abcc12 slc35g3 slc35g5 and encode proteins of unknown drug target type suchas ac0080733 ac1135541 aknad1 al0496342dnlz ervw1 etda fbxw10 fer1l5 lmntd1lrrc72 nms prr23a prr23b prr23c pxt1rfpl4b and ssx4b many of these genes are listed infigs andor fam236a figs and and obp2b met candidatethreshold through our analysis of human testes datasetsbut did not meet candidate threshold from any of thegerm cell or sertoli cell datasets indicating potential expression in peritubular myoid cells leydig cells or othercell outside of the seminiferous epithelium fam36a hasnot been previously identified and neither mouse orthologs 1700011m02rik gm9112 have been knocked outobp2b was previously identified through djureinovic and johnston however of the equivalent mouse orthologs lcn4 obp2a obp2b only obp2ahas been knocked out revealing abnormal coathair pigmentation ism2 and magec2 were identified through both human sertoli cell datasets while also identified throughtestis andor germ cell datasets both genes have beenpreviously identified ism2 magec2 ism2knockout mice display nonreproductive phenotypes consistent with this finding our mouse data do notidentify ism2 as reproductive tractspecific in micemagec2 lacks a mouse ortholog for functional analysis in micehuman sertoli cellspecifickrtap23 krtap412 lhx9 and psg5 were identified through one or both human sertoli cell datasets butwere not identified through any of the testis or germ cell 0crobertson bmc biology page of datasets indicating sertoli cellspecific expression in thetestes additional file table s5 none of these geneshave been previously identified as reproductive tractspecific in humans although lhx9 and psg5 havemouse orthologs that have been knocked out [“]human krtap23 has mouse orthologs krtap52gm4559 gm40460 and gm45618 and human krtap412 has mouse orthologs krtap47 and gm11555none of these mouse orthologs have been knocked outfig and additional file table s5knockin mousepsg5 knockout mice display nonreproductive phenotypes [“] however lhx9 knockout mice display absent testes and sterility due to an essential requirementfor lhx9 during mouse gonad formation a lhx9gfpcreerbyknockingin gfpcreer at the endogenous lhx9 locus”crossed with the rosa26tdtomato reporter mouse linerevealed cre recombinase activity in retinal amacrinecells developing limbstestis hippocampal neuronsthalamic neurons and cerebellar neurons thuslhx9 is not reproductive tractspecific in mice ourmouse data confirm this findingline”generatedhuman epididymisspecificto the

Hydrophobicity drives receptormediateduptake of heatprocessed proteins by THP1macrophages and dendritic cells but notcytokine responsesYing Deng12 Coen Govers1 Malgorzata Teodorowicz3 Ieva Liobyte12 Ilaria DeSimone13 Kasper Hettinga4 Harry J WichersID12 Food and Biobased Research Wageningen University and Research Wageningen The Netherlands Laboratory of Food Chemistry Wageningen University and Research Wageningen The Netherlands Cell Biology and Immunology Wageningen University and Research Wageningen The Netherlands Food Quality and Design Wageningen University and Research Wageningen The Netherlands harrywicherswurnlAbstractAlthough an impact of processing on immunogenicity of food proteins has clearly been demonstrated the underlying mechanisms are still unclear We applied different processingmethods wet heating ˚C and low or hightemperature ˚C or ˚C respectivelydryheating in absence or presence of reducing sugars to lactoglobulin BLG lysozymeand thyroglobulin which represent dietary proteins with different pI or molecular weightUptake of the soluble fraction of the samples was tested in two types of genetically homogeneous antigenpresenting cells macrophages and dendritic cells derived from THP1monocytes This revealed a strong correlation between the uptake of the different proteinsamples by macrophages and dendritic cells and confirmed the key role of hydrophobicityover aggregation in determining the uptake Several uptake routes were shown to contribute to the uptake of BLG by macrophages However cytokine responses following exposureof macrophages to BLG samples were not related to the levels of uptake Together ourresults demonstrate that heattreatmentinduced increased hydrophobicity is the prime driving factor in uptake but not in cytokine production by THP1 macrophagesIntroductionDietary proteins play an important physiological role not only in providing amino acids andenergy but also for the maturation and regulation of the immune system Proteins are knownto be involved in the regulation of chronic inflammation and be the cause of allergies [] Forexample a higher intake of animal protein was found to enhance the proinflammatoryresponse of macrophages in mice [] Most of the proteins that humans ingest have beenthrough a heating process as part of the food processing as a result of which structural modifications such as glycation with reducing sugar andor aggregation of proteins may occura1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Deng Y Govers C Teodorowicz MLiobyte I De Simone I Hettinga K Hydrophobicity drives receptormediated uptake ofheatprocessed proteins by THP1 macrophagesand dendritic cells but not cytokine responses e0236212 101371journalpone0236212Editor Yi Cao Xiangtan University CHINAReceived June Accepted August Published August Copyright Deng This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This work was financial supported by theChina Scholarship Council No awarded to YDCompeting interests The authors have declaredthat no competing interests existPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseDifferent processing methods would alter the immunogenicity of food protein in differentmanner For example the antigenicity of prawn allergen was found to be significantlyincreased after frying but decreased after boiling acid treatment or highpressure processing[] The allergenicity of milk protein was reported to decrease after glycation []In an earlier study on lactoglobulin BLG the major whey protein of milk [] weobserved that heat processing under different conditions altered the physicochemical properties and its uptake by THP1 macrophages In particular heating in solution at ˚C for days introduced changes in hydrophobicity and molecular weight which were shown to be thekey determinants for uptake This event may be relevant for a possible subsequent immuneresponse [ ] and has been linked to heattreatmentinduced changes in protein™s pr sityto induce an allergic reaction [“] Despite the new findings in that study there are obviouslimitations relate to the use of a single protein cell phenotype and immunological readoutHere we extend previous findings by investigating other proteins lysozyme and thyroglobulin another cell phenotype THP1 dendritic cells and different immunologicalresponses to yield novel insight Lysozyme and thyroglobulin were subjected to the same heattreatments as used for BLG Lysozyme from chicken egg white is a single chain polypeptidewith four disulphide bridges and is a rather inflexible protein with stable structure and properties [] It has a molecular weight of kDa which is similar to kDa of BLG and an isoelectric point pI of [] which is clearly different from BLG pI [] resulting in apositive net charge of this protein at the physiological pH Moreover it is also known as anallergen as exemplified by the high incidence of sensitization and allergenicity to this protein[] On the other hand bovine thyroglobulin which originates from follicular cells of the thyroid gland is a protein that is considered as less allergenic It has a molecular weight of kDa as a monomer making it more than times larger than the mass of the other two proteins under investigation It has a complex quaternary structure composed of four subunitsthat are disulphide bonded [ ] Its pI is close to that of BLG conferring it a similarcharge as BLG at the pH of the exposure medium We analysed uptake of heattreated proteinsby macrophages and dendritic cells and correlated this to physicochemical characteristics Furthermore we investigated the mechanism and receptors that are involved in uptake and identified downstream immunological responses by quantifying cytokine responseTaken together our findings provide insights into the role of protein size and pI towardsphysicochemical changes upon foodprocessing related heattreatments This appears to yieldgeneralisable correlations between a protein™s physicochemical characteristics and its uptakeby antigenpresenting cells APCs Finally we identified routes of uptake and downstreamimmunological responses which will help to clarify the interaction of protein ps withmacrophages and the potential immunoregulatory effect The results of this study could helpto better understand how the immunogenicity of food proteins can be modulated throughprocessingMaterials and methodsChemicalsAll chemicals were purchased from Sigma Aldrich St Louis Missouri USA unless otherwisestatedSample preparation and fluorescent labellingLactoglobulin BLG from cow™s milk lysozyme from chicken egg white and thyroglobulinfrom bovine thyroid were dissolved in sodium phosphate buffer mM pH same belowto a protein concentration of mgmL Besides this solution without saccharides DglucosePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseglu Dlactose lac and galactooligosaccharide GOS Royal FrieslandCampina Wageningen the Netherlands were added to reach a final molar ratio of total free amino groups tosaccharide reducing ends Identical heating methods being hightemperature dryheating Hwetheating W and lowtemperature dryheating L sample preparation and naming wereapplied to the proteins as indicated in a previous study [] Fluorescein isothiocyanate isomer IFITC labelling was performed as described previously []THP1 cell culture and differentiationThe human monocytic leukaemia cell line THP1 ATCC Manassas Virginia USA was differentiated into cells characteristic for resting macrophages M0 as described elsewhere []at a concentration of — cellsmL using ngmL phorbol12myristate13acetate PMAfor a hours stimulation followed by washing two times with medium and another hoursof rest Using a cell concentration of — cellsmL and incubation with ngmL of IL4and ngmL of PMA for days as described [] immature dendritic cells were generatedfrom THP1 monocytesPhysicochemical analysisThe analysis of solubility protein mass remaining in solution loss of amino group OPAmethod AGE formation fluorescent measurement exposure of hydrophobicity regionANS method surface charge zetapotential measurement secondary structure circulardichroism measurement and aggregation size exclusion chromatography were performedusing the protocols as described previously []Uptake and blocking of different uptake routesUptake experiments were performed as described previously [] No significant difference ofabsolute uptake value has been found for native BLG thyroglobulin or lysozyme in macrophages or dendritic cells S1 Fig As there is no bias for the basic uptake capability for the different proteins all the uptake results were presented relative to their corresponding nativeprotein to be able to compare among different proteins To determine which route of uptakewas involved in a protein™s uptake by THP1derived cells the cells were preincubated for minutes with DMSO control or different inhibitors μgmL nystatin caveolaedependentuptake inhibitor μgmL chlorpromazine clathrindependent uptake inhibitor μgmLcytochalasin B microphagocytosis inhibitor or all inhibitors combined dissolved in DMSOFollowing incubation the cells were washed with PBS and incubated for hours with FITClabelled protein samples Subsequently the cells were harvested and measured using flowcytometry as described previously [] For all uptake experiments μl trypan blue was addedper μl of cell suspension to quench extracellular FITC signal before flow cytometry analysis Uptake was calculated as either the fold change in mean fluorescence intensity MFI relative to control after correcting for FITC labelling efficiency control or as percentage tocontrol control Soluble Receptor for Advanced Glycation End Products sRAGE CD36and galectin3 inhibition ELISAThe experiment was performed according to the method of Liu [] As a positive control soy protein Bulk Powder Colchester UK was mixed with Dglucose in an equal wwratio in mM PBS buffer pH to a protein concentration of mgmL and heated for minutes at ˚C The positive control was diluted in mM of sodium carbonate buffer pHPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine response to μgml and used for plate coating by adding μL per well to a Nunc MaxiSorp„¢ flatbottom plate Thermo Fisher Waltham Massachusetts USA and incubating overnight at ˚C Then the plate was washed times with PBS buffer pH containing vv Tween After blocking with bovine serum albumin BSA in PBS for hour at room temperature the wells were washed times and incubated with μL protein samples μgmL at ˚C for hour The samples were preheated at ˚C for minutes with recombinanthuman sRAGE CD36 or galectin3 at a concentration of or μgmL RD SystemsMinneapolis Minnesota United States in PBS buffer with BSA and Tween20For detection the plate was washed times and incubated with μL per well of monoclonalmouse IgG2B human sRAGE or galectin3 antibody RD Systems Minneapolis MinnesotaUnited States at a concentration of μgml for minutes shaking at room temperatureAfter washing times the plate was incubated for minutes while shaking at room temperature with μL per well polyclonal goat antimouse HRPconjugated DAKO Glostrup Denmark at a concentration μgmL For galectin3 an additional incubation step with μgml Streptavidin SDT Baesweiler Germany μL per well for minutes was followed For CD36 only incubation with goat antihuman IgGHRP SouthernBiotech Birmingham Alabama United States at a concentration of μgml for minutes shaking atroom temperature was used for the detection step For all receptor inhibition ELISAs the platewas then washed times and μL per well TMB substrate was added and incubated for minutes before stopping the reaction by adding uL of HCl per well The absorbancemeasured at nm by an Infinite1 PRO NanoQuant Tecan Ma¨nnedorf Switzerlandwas subtracted from the measured absorbance at nm Each sample was measured in triplicate and values were averaged Absorbance of buffer was used as a control and was subtractedfrom every sample The absorbance of the control solution without any inhibition was codedby Abscontrol The percentage of inhibition for samples was equal to AbscontrolAbssampleAbscontrol�Cytokine production measurementTHP1 macrophages were incubated with μgmL of protein sample or medium as controlfor hours after which the supernatant was collected The cytokine production was measured using the ELISA Deluxe Set Human IL8CCL20IL1 Biolegend San Diego California United States following the manufacturer™s protocolStatisticsThe statistical analysis was performed using Prism software GraphPad Software San DiegoCalifornia United States with p considered to be significant The correlation analysiswas done by calculating the Pearson correlation coefficient r and the twotailed p value ThePCA plot was generated with the dataset of variables after being mean centred and weightedby 1standard deviation using Unscrambler software CAMO Oslo NorwayResultsWetheating significantly increased uptake of thyroglobulin by THP1derived macrophages whereas heattreatment did not increase uptake oflysozymeThyroglobulin and lysozyme were wetheated W hightemperature dryheated H or lowtemperature dryheated L in the absence or presence of saccharides with different lengthsmonosaccharide glucose disaccharide lactose or oligosaccharide GOS Soluble proteinPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsefrom each sample was concentration calibrated fluorescently labelled and incubated with resting macrophages that were derived from THP1 monocytes Hightemperature dryheating ofthyroglobulin in the presence of glucose and lactose and lysozyme in the presence of glucoseled to complete insolubility S2A and S4A Figs and therefore these samples were excludedfrom further analysis Prior to the experiment the lipopolysaccharide LPS contaminationwas measured S1 Table and found to be below the threshold level that significantly affecteduptake capacity of THP1 macrophages ie ngmL as reported in an earlier study []For uptake of thyroglobulin samples by macrophages Fig 1A the type of heat treatmentappeared to play a more determining role than the presence or absence of saccharides Notably all wetheated samples regardless of the presence of saccharides showed significantlyincreased uptake compared to untreated protein Although not significantly differing fromuntreated protein high and lowtemperature dryheating increased the uptake of thyroglobulin as well On the contrary no significant differences in uptake of lysozyme after differenttreatments and saccharide exposures was noticeable Fig 1BUptake of heattreated protein samples by THP1derived immaturedendritic cells is qualitatively similar to that of THP1derivedmacrophagesThe uptake of processed thyroglobulin and lysozyme as well as BLG which was previouslyreported [] was only tested for macrophages To expand the understanding of the proteins™uptake beyond only macrophages THP1derived immature dendritic cells iDC were alsotested with identical methodology as for macrophages The uptake of wetheated thyroglobulinin the absence or presence of saccharides by these iDC was significantly increased compared tothe native protein Fig 2A For lysozyme the relative uptake remained the same regardless ofthe treatment Fig 2B The uptake of wetheated BLG in the absence or presence of saccharidesby iDCs was also increased compared to the native form although only reaching significanceFig Uptake of thyroglobulin by macrophages was significantly increased upon wetheating of the sample Thyroglobulin THY A and lysozymeLYS B were untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS THP1derived macrophages Mϕ wereincubated with FITClabelled soluble fraction of protein samples for hours after which uptake was determined using flow cytometry The resultsrepresent mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences comparedto native proteins were calculated with Dunnett™s Test �p ��p 101371journalpone0236212g001PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin lysozyme and BLG samples by THP1derived iDC was strongly and significantly correlated with uptake bymacrophages AC Thyroglobulin THY lysozyme LYS and BLG were untreated or heated W H or L in the absence or presence of saccharidesglu lac or GOS THP1derived iDC were incubated with FITClabelled soluble fraction of protein samples for hours after which uptake wasdetermined using flow cytometry The results represent the mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences compared to native proteins were calculated with Dunnett™s Test ��p ���p D Therelative uptake values of all protein samples by iDC was plotted against macrophages Mϕ and the correlation analysis was done by calculating thePearson correlation coefficient r and twotailed p value101371journalpone0236212g002when heattreated in the presence of glucose Fig 2C When compared to the results obtainedwith macrophages the increase in uptake upon protein wetheating for iDC was less pronounced BLGmacrophage data from previous study [] However the uptake pattern of allsamples correlated strongly r p between the two cell types Fig 2DIncreased uptake of thyroglobulin by THP1derived macrophages wascorrelated to hydrophobicity and aggregationDue to the strongly correlated responses of iDC and macrophages in uptake with a higher relative uptake by macrophages the latter was used for further testing To explain thePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsephysicochemical mechanism behind the differing uptake capability of macrophages for differently treated thyroglobulin and lysozyme a set of measurements was performed on the solublefraction after treatmentFor thyroglobulin dryheating at hightemperature significantly decreased its solubilityresulting in the absence of a soluble fraction when heating was performed in the presence ofglucose and lactose S2A Fig The soluble fraction of samples in the presence of GOS or without saccharide revealed a strong decrease of free amino groups and increases in AGErelatedfluorescence and formation of polymers in the presence of GOS S2B S2C and S3 Figs Wetheating did not result in loss of solubility but also led to formation of polymers and albeit lesspronounced as for hightemperature dryheating significant losses of amino groups andincreases in AGErelated fluorescence Moreover wetheating induced a significant increasein the exposure of hydrophobic regions as measured by ANSbinding S2D Fig Dryheatingat lowtemperature did not have much influence on the measured parameters for thyroglobulin except for a significant loss of amino groups and a slight increase in oligomer and polymerformation S2B and S3 Figs There was no significant change in the secondary structure forany of the treated thyroglobulin samples S2E and S2F FigApplying identical heating and glycation conditions as for thyroglobulin did not generateany soluble aggregates for lysozyme checked by size exclusion chromatography Hightemperature dryheating led to a significant decrease of solubility with complete insolubility forthe samples heated in the presence of glucose S4A Fig Among the soluble fraction of thehightemperature dryheated samples there was a significant decrease of free amino groupsincreases of AGErelated fluorescence and exposure of hydrophobic regions in the presence ofsaccharides S4B“S4D Fig Moreover results showed a significant loss of sheet structure forall samples except when heated in the presence of GOS S4F Fig Similarly lowtemperaturedryheating resulted in a significant loss of sheets in all lysozyme samples For wetheatedlysozyme only the exposure of hydrophobic regions in the presence of saccharides was significantly increased S4D FigTo identify the most important physicochemical properties of heatprocessed thyroglobulinthat are related to its uptake by macrophages we performed a principle component analysisPCA and a correlation analysis In the PCA plot Fig all lowtemperature dryheated samples clustered together with the untreated thyroglobulin indicating that the physicochemicalproperties of these samples were similar Uptake was strongly related with wetheating andpositively related to hydrophobicity ANS and polymer proportion and inversely to monomer and oligomer proportion These relations were further confirmed by correlation analysesAs shown in Fig there was a strong correlation between uptake and hydrophobicity and amoderate correlation between uptake and aggregation as indicated by the fraction of monomers polymers and oligomers Of note is that the samples were strongly clustering into twogroups at the extremes of the data scale for both the uptake and monomer or polymer contentcorrelation plots Fig 4B and 4C the correlation between uptake and oligomer content wasless profound Fig 4D p Contrastingly to thyroglobulin no clear correlation could befound between treatment and physicochemical properties for lysozyme in the PCA plotS5 FigEDCmediated increase in hydrophobicity but not crosslinking is themain factor driving uptake by macrophagesEDC links the carboxyl and amino group of amino acids and thereby offers the possibility tocrosslink proteins without heating This allows for a discrimination between aggregation andother heatingrelated effects like hydrophobicity changes with regard to their impact onPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin by macrophages is related to wetheating hydrophobicity and aggregation Thyroglobulin THYwas untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS and tested for solubility uptake ofsoluble fraction by THP1 macrophages uptake AGE formation AGE glycation OPA percentage of αhelix helix or sheetsheet aggregation monomer and smaller mono oligomers oligo polymers poly and exposure of hydrophobic regions ANSPCA scores of the samples were given in blue and the parameter loadings of the principal components were given in red The clusteringof uptake with wetheated samples the formation of hydrophobic regions and polymer proportion is indicated in the red circle101371journalpone0236212g003uptake As shown in Fig 5A and 5B the proportion of polymer and monomer significantlyincreased resp decreased in the EDC crosslinked BLG and thyroglobulin On the contraryonly limited aggregation of lysozyme was observed upon EDCtreatment EDC crosslinkedBLG and thyroglobulin did not demonstrate an increase in uptake Surprisingly EDCmediated crosslinking significantly increased the hydrophobicity of lysozyme Fig 5C and also itsuptake by macrophages Fig 5DUptake of heatedtreated BLG was mediated via several routesTo better understand the mechanism of uptake we set out to identify which routes of uptakewere utilized for the various proteins that differed in physicochemical characteristics andtherefore in their recognition motifs for cells Native and heattreated BLG in the presence ofglucose was used as a subset because these samples covered the whole range of levels of physicochemical modifications and macrophages were pretreated with inhibitors targeting phagocytosis caveolae or clathrinmediated uptake or a combination thereof Preincubation ofmacrophages with nystatin caveolae inhibitor and the combination of inhibitors prior toBLG samples exposure significantly inhibited the uptake Fig The usage of cytochalasinmicrophagocytosis inhibitor significantly decreased the uptake of both hightemperaturedryheated and wetheated BLG Fig 6B and 6C Moreover chlorpromazine significantlyPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Correlation analysis of uptake of heattreated thyroglobulin with physicochemical parameters Uptake of soluble fraction ofthyroglobulin THY samples by macrophages was correlated to hydrophobicity r07 and proportion of monomer polymer and oligomer07r05 The correlation analysis was done by calculating the Pearson correlation coefficient r and twotailed p value101371journalpone0236212g004reduced uptake of wetheated BLG indicating that clathrin was involved in this processFig 6CInhibition ELISA showed high binding of hightemperature dryheated andwetheated BLG by sRAGE CD36 and galectin3We investigated several receptors that might be involved in binding and subsequent uptake ofthe BLG samples that were heattreated in the presence of glucose Using an inhibition ELISAthe binding of the BLG samples to the soluble receptor for advanced glycation end productssRAGE CD36scavenger receptor and galectin3 was analysed Fig Native BLG did notshow any binding to sRAGE galectin3 or CD36 In contrast hightemperature dryheating ofBLG induced significant receptor binding for all three tested receptors Wetheating of BLGsimilarly resulted in binding to all three receptors albeit to a lesser extent and only significantly for sRAGE and galectin3 Finally lowtemperature dryheating did not induce anyreceptorbindingPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig EDCmediated hydrophobicity but not aggregation resulted in increased uptake of proteins by THP1 macrophagesThyroglobulin THY lysozyme LYS and BLG were nontreated or treated with EDC and subsequently analysed for the proportion ofpolymer A the proportion of monomerdimer B and protein hydrophobicity C EDCtreated and nontreated proteins were fluorescentlylabelled and their uptake by macrophage Mϕ was measured D The results represent the mean values ± SD of “ independent experimentswith independent sample sets Statistical differences were calculated with twotailed unpaired Ttest with Welch™s correction �p ��p ���p 101371journalpone0236212g005Cytokine responses of macrophages following exposure to BLG werereduced upon heattreatment of BLGReceptor binding and uptake of protein samples can lead to immune activity The immunological response could be estimated by the expression of secreted cytokines or chemokines Uponexposure to medium or native or heattreated BLG in the presence of glucose the response ofmacrophages was analysed by measuring the levels of secreted IL8 CCL20 and IL1 Asshown in Fig nonprocessed native BLG and lowtemperature dryheated BLG induced significant levels of IL8 CCL20 and IL1 secretion in macrophages when compared to mediumPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Several routes appeared to be involved in the uptake of wetheated BLG BLG was untreated or heated W H or L in the presence of glucose gluand the soluble fraction was FITClabelled Macrophages were pretreated with DMSO control or inhibitors dissolved in DMSO of clathrindependentuptake chlorpromazine chlor caveolaedependent uptake nystatin nys microphagocytosis cytochalasin cyto or a combination of them combinedfor min before a 2hour incubation with protein samples Protein uptake was subsequently measured using flow cytometry The relative MFI wascalculated by dividing the MFI of a sample by the MFI of the corresponding control The results represent mean values ± SD of multiple independent cellexperiments N “ with independent sample sets The statistical differences were calculated with onetailed unpaired Ttest compared to control�p ��p ���p 101371journalpone0236212g006Hightemperature dryheating of BLG significantly reduced the secretion of all three analysedcytokines when compared to native BLG A similar reduction was observed in wetheated BLGfor IL8 and IL1 but less strongly and not significantly for CCL20DiscussionIn an earlier study we observed that hydrophobicity and the aggregation state of heatprocessed BLG were important parameters that were related to the protein uptake by THP1PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Heattreatment in the presence of glucose confers receptor binding capacity to BLG BLG was untreated orheated W H or L in the presence of glucose glu and the soluble fraction was used to inhibit the binding of receptorto a positive control in the inhibition ELISA Data shown are the mean values ± SD of n based on measurement of parallel prepared samples Statistically significant differences between native and processed BLG for each receptorwas determined using unpaired twotailed tTest �p ��p ���p 101371journalpone0236212g007macrophages [] Here we extended these findings by analysing whether similar relationswould apply for a protein with similar pI but higher molecular weight thyroglobulin aprotein with similar molecular weight but higher pI lysozyme EDCaggregated proteinsand another cell type by using THP1 dendritic cells The comparison between THP1derived macrophages and dendritic cells regarding uptake revealed similar patterns for thethree tested proteins and the different heattreatments Macrophages and DCs are both able tocapture exogenous ps and induce an immunogenic response playing an essential role inFig Cytokine and chemokine responses of macrophages upon exposure to native or heattreated BLG varies BLG was untreated or heated W Hor L in the presence of glucose and the soluble fraction was incubated with macrophages The cytokine secretion of IL8 A CCL20 B and IL1 Cwas analysed in the supernatant with ELISA The results represent mean values ± SD of “ independent experimental measurements The statisticaldifferences were calculated with Tukey™s Test with different letters a“c in the bars indicated significantly different p 101371journalpone0236212g008PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseimmunological defence [] We did observe however a quantitative difference in uptake asthe uptake values for wetheated thyroglobulin and BLG were relatively higher in macrophagesthan in DCs This suggests that macrophages are more efficient in their phagocytic role thanDCs which is in line with literature stating that uptake by macrophages is part of their corerole in eliminating harmful substances whereas uptake by DCs is a means to initiating anadaptive immune response [] Upon comparing the three tested proteins we found that thephysicochemical alterations upon heattreatment to thyroglobulin and BLG were similar butdifferent from lysozyme These proteins differ in size thyroglobulin kDa BLG kDa lysozyme kDa pI lysozyme BLG thyroglobulin anddenaturation temperature lysozyme ˚C BLG ˚C thyroglobulin ˚C [“]suggesting that both pI and heat stability may be important for heatinduced structural proteinchanges The high pI may have conferred a positive charge to lysozyme during wetheatingpH thereby hindering aggregation under this condition due to electrostatic repulsionThe strong internal coherence and stability of lysozyme™s structure may also have contributedto its stability during heat processing [] It is reported

pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airflow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue fibroblasts To date no studiesare available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial fibroblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDfibroblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSfibroblasts p � and p � for TGF1 at h in CSfibroblasts p � for procollagen1 at h inCOPDfibroblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects Backgrounde progressive chronic airflow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ‚ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [“] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myofibroblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial fibroblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic fluid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac fibroblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial fibroblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici ScientificiMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial fibroblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial fibroblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until confluentprimary fibroblasts were obtained 16HBE cells and primarybronchial fibroblasts were maintained in Dulbecco™s modified minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were “ confluent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to × cellsmL were placed in mm polypropylene tubes completely filled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterfilled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy flux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary fibroblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated fibroblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantification of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was purified and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturer™sinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at ˆ’°CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast„¢ SYBR Green RTPCR kitITaccording to the manufacturer™s instructions and the genespecific primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Briefly the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step amplificationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the specificity of PCR amplificationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ˆ’ΔCt method[] for primary bronchial fibroblasts and the ˆ’ΔΔCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantification in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturer™s instructions Table CKit CD117PCNA and NFκBp65 were quantified in cell lysates CD90TGF1 and procollagen1 were quantified in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse anti“thyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identification of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspecificmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial fibroblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialfibroblasts for œin vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMM”MMM”± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDND”FEV1FVCCS1CS2CS3Ex””capacity ND not determined Patients were classified according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientific ngmL “ ngmL ngmL “ ngmL pgmL “ pgmL pgmL “ pgmL pgmL “ pgmL17pgmL “ pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial fibroblasts Figure 1a ESWtreated primary bronchial fibroblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialfibroblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial fibroblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSfibroblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDfibroblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial fibroblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial fibroblasts from control smokers CSbronchialfibroblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 3a“3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialfibroblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cellscompared with nontreated cells Figures 4a“4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial fibroblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial fibroblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared withnontreated cells Figures 5a“5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial fibroblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial fibroblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cells compared with nontreated cellsFigures 6a“6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestˆ—ˆ—p andˆ—ˆ—ˆ—p Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial fibroblastsof COPD patients n � a primary bronchial fibroblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial fibroblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial fibroblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 7a“7c NFκBp65 protein in the cell lysates was decreased in primary bronchial fibroblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial fibroblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identified by the use ofanti“thyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identification of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial fibroblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial fibroblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—CST0T24T48T72NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—16HBET0T24T48NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ— 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial fibroblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialfibroblasts of COPD patients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 “ˆ† Ct0180021103960767CS CNCS ESWcKit CD117 “ˆ† CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 “ˆ†ˆ† CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA “ˆ† CtCS CNCS ESWT0T24T48000510152025PCNA “ˆ†ˆ† Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CS“primary bronchial fibroblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CS“primary bronchial fibroblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPD“primary bronchialfibroblasts after ESW treatment A marker of ‚ammationtranscription factor NFκBp65 protein was decreased inCOPD“primary bronchial fibroblasts at h after ESWtreatment but it was increased in CS“primary bronchialfibroblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 “ˆ† Ct0005101520CS CNCS ESWThy1 CD90 “ˆ† CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 “ˆ† ˆ†CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patients ad primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 “ˆ† Ct00051015CS CNCS ESWT0T24T48TGF 1 “ˆ† Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 “ˆ† Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 “ˆ† Ct000510152025CS CNCS ESWT0T24T48Procollagen1 “ˆ†ˆ† Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patientsa d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial fibroblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial fibroblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[“] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial fibroblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon fibroblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial fibroblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPD“primary bronchial fibroblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation confirming again the proproliferative role of ESW exposurefor these lung structure cells is finding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CS“primary bronchial fibroblasts was notobserved in ESWtreated COPD“primary bronchial fibroblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CS“primary bronchial fibroblastsafter ESW treatment but not in COPD“primary bronchialfibroblasts ese diï¬erences in the response to ESWchallenge of COPD“ and CS“primary bronchial fibroblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedfibroblasts we interpret the increment of y1 protein NFκBp65 “ˆ† Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 “ˆ† Ct01234T24T48T72T0CS CNCS ESW NFκBp65 “ˆ†ˆ† Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatment”like that of cKit”as a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCS“primary bronchial fibroblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialfibroblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary fibroblasts[] even though it was also reported that the increase ofprofibrotic markersincluding procollagen1 in humanlung fibroblasts may be NLRP3 ‚ammasome dependentand TGF independent [] and associated with increased‚ammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPD“primary bronchial fibroblasts at h after ESW

" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the ˜trial within cohorts™ twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio„¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately “ disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act [“]despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [“] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually“ bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [“] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [“] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the ˜trialdifferent from the conventional rctwithin cohorts™ twics design [“] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection “ days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ‰¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and “ days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately “ will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within “ days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio„¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio„¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including “ monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within“ years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscox™s proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator “ together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of ˜disappointment bias™ in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participant™s eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para

" while the impact of family caregiving has been welldocumented many of such studies center oninvestigating external factors such as socioeconomic status accessibility to resources and availability of socialsupport as the primary causation of caregiver wellbeing outcomes this paper explores the motivations that drivefamily caregivers in supporting their family members at the endoflife and critically examines how internalappraisal processes of such motivations can both positively and negatively impact their wellbeingmethods this study adopted an interpretative phenomenological analysis ipa to investigate the motivations andinternal appraisal processes of asian family caregivers in singapore who were tending to a dying family memberqualitative dyadic interview data n was drawn from a larger randomized controlled trial for a novel familydignity intervention fdi for palliative care patients and their families the sampling population consisted ofparticipants aged and above who were identified to be the primary caregivers of older palliative care patientswith a prognosis of less than months data collection was conducted in the homes of patients and familycaregiverscontinued on next page correspondence andyhyhontuedusg1psychology programme school of social sciences nanyang technologicaluniversity singapore singapore2centre for population health sciences lee kong chian school of medicinenanyang technological university singapore singaporefull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctanho bmc palliative care page of continued from previous pageresults findings revealed six themes that could either nurture or diminish caregiver wellbeing honoringfidelity caregivers were motivated to commit to their caregiving roles in order to avoid regret alleviatingsuffering caregivers were motivated to relieve their family member™s pain enduring attachment caregiverswere motivated to spend time together with their family member preserving gratitude caregivers weremotivated to express their appreciation to their family member by caregiving navigating change caregiverswere motivated to adapt accordingly to changes in the illness trajectory and reconciling with mortalitycaregivers were motivated to respond accordingly to their family member™s prognosis the final theme of thewellbeing determinant is posited as an indication of selfdetermination and is conjectured to influence howcaregiving motivations are appraised by the caregiver fulfilling and enhancing one™s sense of selfdetermination appears central to infusing one™s caregivingmotivations with positive meaning and consequently nurturing one™s wellbeing in the endoflife caregivingjourney these findings are discussed with recommendations for healthcare professionals working with familycaregivers of palliative care patientskeywords palliative care caregiver motivations wellbeing meaning burnout resilience selfdetermination endoflife qualitative research the experience of an endoflife eol family caregivercan be likened to a paradox “ what could evoke a senseof pleasure appreciation and gratitude could also bringabout feelings of anxiety distress and pain while somehave related the caregiving journey to the metaphor ofascending a mountain the expedition of an eol family caregiver usually spans beyond merely a couple ofdays weeks or months they must navigate the peaks ofdiagnosis to prognosis and eventually death and bereavement in what often unfolds into a lifelong climbthe role of the eol family caregiver is often multifaceted and interminable daily duties involve managingmedical regimes traversing the healthcare system andtaking charge of other dependents alongside providingphysical mental and emotional support throughout theillness trajectory [ ] many family caregivers are rarelyequipped with formal or adequate training and nor dothey possess sufficient resources and skills before theyfind themselves embroiled in eol caregiving responsibilities family caregivers must also process and manage amultitude of thoughts and emotions as they come toterms with the changes and sometimes losses in theirpersonal lives this complex experience is not limited to a minorityof people despite strong global advancements in medical technology and healthcare systems older populations remain highly susceptible to chronic and terminalmorbidities that are incapacitating in the unitedstates alone over million caregivers tend to their ailing family members annually while an estimated of patients in europe requiring longterm care areattended to by informal caregivers with the anticipated number of older adults in the world soaring to billion by the year there will certainly be a surging demand for family caregivers to relieve the ensuingresource strains on healthcare settingsthe impact of family caregiving stressors has beenwelldocumented [“] with various studies exploringcaregiver burnout and its effects on the community andsociety complementing these studies are literature thatreveal characteristics of resilience and transformationalgrowth displayed by family caregivers in adversity [“] the common thread that impacts both caregiverburnout and resilience appears to be a lack of or adequate coping a process that requires the individual toconstantly change efforts in both thoughts and behaviorsin order to manage internal or external demands thatare considered stressful despite this indication thatone™s psychological resources are key to maintainingone™s wellbeing many studies often consider externalfactors such as socioeconomic status accessibility toresources and availability of social support as the primary causation for the degree of caregiver wellbeingthus these studies often recommend pragmatic interventions that focus on improving external circumstancesaccordinglyperception emotion and motivation of the familycaregiverwhile it is undoubtedly beneficial to mitigate tangiblestressors one must not lose sight of the magnitude of aperson™sthedemands of caregiving this perception and appraisalembody one™s subjective caregiver burden “ observed inreviews of over caregiver studies to pose deepseatedimplications on caregivers™ quality oflevels ofdepression and anxiety and stress coping [ ]internal perception and appraisal oflife 0ctanho bmc palliative care page of campbell later confirmed this observation in astudy that evaluated multiple variations in the caregiverexperience subjective caregiver burden was repeatedlyidentified as the primary indicator for caregiver stressit was around the same time that folkman andmoskowitz discovered that caregivers experiencepositive emotions alongside negative emotions duringstressful events they put forth the tenet of meaningfocused coping in which caregivers derive mental andemotional sustenance thus effecting positive emotionsin challenging circumstances by deferring to their beliefsvalues and existential goals folkman and moskowitzfound that meaningfocused coping is an intrapsychicprocess of discovering benefits in caregiving reminding oneself of such benefits setting goals thatinspire a sense of mastery and competence realigningpriorities in view of changes and infusing ordinaryevents with positive meaning folkman furtherattested that meaningfocused coping exists alongsidenegative appraisals in a caregiver™s stress process inorderand psychologicalresources during stressful eventsto reinstate physiologicalgiven the importance of psychological appraisal it isreasonable to postulate that gaining insight into a caregiver™s beliefs values and goals that stimulate them incaregiving defined as motivations in this paper wouldyield valuable information that could be used to enhancemeaningfocused coping such interventions would target the essence of a person™s selfconcept “ that is theindividual™s perception of who they are and what theybelieve in “ and transcend current cursory social mentaland emotionalfor caregiverstresssymptom management[ ] a studybridging the research gap in asian caregivingwhile the multidimensional nature of caregiving burdenand coping is not confined to any specific culture thereare distinctive elements that bear influence on the asiancaregiving experience family takes precedence in asiansocieties with strongly inculcated values and expectations of filial piety and filial responsibility placed uponfamily membersconducted insingapore a multiethnic society that is predominantlychinese found that family caregivers who internalizedand prioritized societal expectations as motivations overtheir personal wellbeing most often faced internalconflict and were highly likely to experience difficultiesin maintaining their mental health familial ties and caregiving duties this is corroborated by the evolvingattitudes towards such confucian rules “ younger asiangenerations no longer perceive absolute submission orcomplete obedience to the family as instrumental valuesin a modern and globalized society and it would bevaluable to further understand how these complexitiesmight manifest in a family caregiver™s motivationsthis paper aims to contribute to and grow the currentbody of knowledge for asian eol family caregivers byanswering the following research questions what arethe internalized motivations defined here as unconone™ssciously assimilated beliefs and valuesattitudes or behaviors of asian family caregivers how might these motivations affect the way they respond to caregiving and impact their wellbeing howcan an understanding of these motivations be integratedinto psychosocial interventions to enhance and sustaincaregiver wellbeingintomethodsresearch design and proceduresthe current study draws qualitative dyadic interviewdata n from a larger randomized controlled trialfor a novel family dignity intervention fdi for asianpalliative care patients and their families n thesampling methods inclusion criteria interview procedure and study protocol for the fdi are comprehensivelydescribed by ho briefly the fdi is developedbased on an integrative body of empirical investigationthat focuses on dignified endoflife care in both western and asian contexts [ ] it integrates elements oflogotherapy and narrative life review to provide psychosociospiritual support to patients and families facingmortality and has been piloted for acceptability andfeasibility before being fully adapted into the intervention study in practice fdi comprises a recorded dyadicsemistructured interview with a patient and a familycaregiver conducted in their homes the fdi therapistuses a guided question framework to facilitate joint conversation on shared memories and living wisdoms thatlead to meaningmaking and the expression of appreciation and reconciliation this is done with the ultimategoal of creating a legacy document that tells the life storyof the patient and is bestowed to the rest of the familythrough an open reading each interview lasted between and min and was conducted in english malaymandarin or a chinese dialect hokkien teochew orcantonese these recorded interviews were transcribedverbatim translated into english by a native languagespeaker where applicable and edited into legacy documents transcripts and legacy documents were reviewedand finalized by patients and caregivers to ensure accuracy and authenticitysamplingthe sample drawn for this study consisted of primaryfamily caregivers of older palliative care patients aged and above with mainly a cancer prognosis of lessthan months eleven were spousal caregivers seven 0ctanho bmc palliative care page of were adultchildren caregivers and two were siblingcaregivers the majority were female aged between to with a mean age of years see table for caregiver demographics they were recruited through theinpatient daycare and homecare hospice service unitsof hca hospice care dover park hospice tan tockseng hospital singapore cancer society and methodistwelfare services the inclusion criteria required familycaregivers to be above years old and identified by thepatient to be their primary carer patients and familycaregivers came from varioussocioeconomic s and were predominantly of chinese ethnicityas the fdi focused on patient narratives transcriptsbearing a moderate to sizeable amount of input from thefamily caregiver were selected for data analysisdata analysisstudy adopted an interpretative phenomenothislogical analysis ipato investigate the internalizedmotivations of family caregivers in tending to a dyingfamily member the ipa is an approach in qualitativeresearch that aims to provide insights into how an individual makes meaning out of a phenomenon itis important to note that as the current study drawsqualitative data from the larger fdi study no explicitinternalized caregiver motivationsquestionsaboutwere asked reflections on motivations towards caregiving occurred anically throughout the interviewtranscripts and were identified by the researcher usingipa through a process of data reduction and datareconstructionfirst authors and screened all transcripts andselected those that had adequate inputfrom familycaregivers to be used for analysis authors and thenconducted linebyline coding to develop descriptivethemes and analytical categories conceptualizing newinterpretation of the data this was followed by regularmeetings among all authors for the further refinement ofthemes and categories to encapsulate the meaning andcontent within the cluster of similar codes with theemergent themes and subthemes created via a summarychart all authors reviewed and defined the emergentthemes once consensus was reached operational definitions were created finally relationships between categoriesthemes and subthemes were proposed andmapped with supporting quotes from transcripts to address issues of trustworthiness and credibility emergentthemes were constantly compared and contrasted withinand across groups during regular meetings the finaltheme categorization and definitions were agreed uponby the entire research team and data saturation and investigator triangulation were achieved table demographics of family caregiversidentifierdph14caregiver relationshipchildcaregiver ethnicitychinesepatient™s diagnosislung cadph19dph34dph42dph53dph59dph68hca12hca68hca75hca81hca87hca109hca114hca116hca117mws004scs18ttsh61ttsh65spousespousesiblingspousechildspousespousechildchildchildspousespousespousespousespousesiblingspousechildchildchinesechinesechinesechinesechinesemalayeurasianchinesemalaychinesemalaychinesechinesechinesechinesechinesechinesemalaychineseprostate calung casigmoid calung cagynaecological malignancylung caprostate cacolon cabreast caendometrial carenal caendometrial cabrain canasopharyngeal capancreas cacopdliver calung cagynaecological capatient™s prognosis months“““““““““ 0ctanho bmc palliative care page of didn™t i do this why didn™t i do that™  dph19spousein some instances family caregivers displayed poignant emotion and dedication to their family membersconveying the great extent to which they would goto give theirthe best care andcomfortfamily membersœi wish to care for him till the very end¦ i want thebest for him and i will do what™s best for him i amwilling to sacrifice my soul to make that happen ortake his place if i could dph68 childalleviating suffering n family caregivers displayed awareness and empathytowards their family member™s physical and emotionalsuffering tied in with a desire to relieve their painœthis morning she was upset with me for forcing herto drink the bitter medicine i told her ˜i love you iwouldn't do this if i had a choice i want you todrink this for your own benefit not mine i'm just encouraging you from thedph53spousesidelines™underlining this motivation to alleviate suffering was thefamily caregivers™innate compassion for their familymember an empathic bond so strong that witnessingtheir family member™s suffering caused them deep emotional distressœ¦ it hurts a lot to drain the fluids i™m heartbrokenwhen i see how much pain she is in especially wheni see the tubing being inserted it must hurt somuch hca109 spousefindingsfigure presents the six caregiving motivations and onewellbeing determinant generated from data to form theblessings or burdens of endoflife caregiving bobecmodelthe six caregiving motivations honoring fidelityalleviating suffering enduring attachment preservinggratitude navigating change and reconciling withmortality represent the beliefs values and goals that areassimilated into the eol family caregiver™s daily life eachmotivation is posited to affect the way a caregiver makesmeaning of their role hence leading to a nurturance ofcaregiver wellbeing termed in this paper as blessings ora diminishment in caregiver wellbeing termed in thispaper as burdens the wellbeing determinant which ischaracterized by the caregiver™s sense of control selfempowerment and kinship derived from their experiences serves as an indication of selfdetermination andis theorized to have a positive influence on how the sixcaregiving motivations are appraised by the caregiverthese themes are described in greater detail and demfrom study participantsonstrated by direct quotesbelowhonoring fidelity number of transcripts theme hasoccurred in n family caregivers expressed their faithfulness and commitment to attend to the needs and wishes of their family members for the remainder of their lives fearingregret to see things through till the end this motivatedthem in fulfilling their sense of duty to the utmost oftheir abilitiesœi shouldn™t regret anything whatever i can do forhim i will do my best and [instead of waiting till]he™s in the coffin you know [and then say] ˜oh whyfig the blessings or burdens of endoflife caregiving bobec model 0ctanho bmc palliative care page of enduring attachment n family caregivers experienced a prevailing attachment totheir family member that motivated them in spendingcherished time together and doing all they could toensure that their family member was well taken care ofœi think i try to make him as comfortable as he canbe every medical checkup every appointment wewill keep to it and i will always be there for him[there will never be] any appointment that i am notgoing with him hca117 spousesome family caregivers found that the motivation tosustain this attachment was also driven by feelings ofanxiety about their family members™ wellbeing thesecaregivers felt the need to be within their family members™ physical proximity as much as possibleœi get worried when she™s lying there and sleepingbecause i™m not sure if anything has happened toher i™m much happier when she™s sitting here withme when she™s just lying there i would think ˜ohno what if something has happened to her™ and i™dbe worried dph59 childpreserving gratitude n family caregivers described past circumstances and beliefs that motivated present feelings of gratitude to theirfamily members this consequently influenced their efforts and responses in caregivingœshe was constipated for as long as a week and shedidn™t tell me when she eventually relieved herselfshe made a mess on the bathroom floor as i wascleaning the mess i thought about how she hadcleaned me up when i was little so i didn™t mindhca81 childwhile many family caregivers reported feelings of gratitude stemming from how their family member hadtreated them in the past some indicated that religiousand cultural beliefs had indoctrinated a sense of indebtedness to their family membersœmy mother says i was indebted to my brother in mypast life this is why i have to settle my debt in thislifetime [by caregiving] because he is here to get hiscompensation mws004 siblingnavigating change n family caregivers reflected on their perceptions of thechanges that had taken place in their lives and that oftheir family members throughout the illness trajectorysome caregivers found motivation in helping their familymembers adaptenergy to liftsupportto changes by dedicating time andtheirspirits and provide emotionalœi would bring my father food when i visit while myhusband would share words of encouragement andtalk to him to cheer him up we just want him to behappy so that he wouldn't spend the whole day innegativity ttsh65 childothers saw the changes as a temporary setback andfound motivation in steering their family member backto their previous condition if possibleœsometimes i will move his legs a little to give himthat exercise i hope that he can walk again but itdepends on how strong his willhca116spouseisreconciling with mortality n the knowledge of their family member™s prognosis motivated some family caregivers to make the most of thetime left with their family members “ creating treasuredmemories and remembering their legaciesœall of us just want to cherish the time that we haveleft with her and we want her to help us spend moregood times together we [want to] learn about mygrandmother learn about my mother so that wecan pass on to the next generation share with themthe traits and the role models to look up tottsh61 childother family caregivers perceived their family member™sprognosis to be unacceptable choosing to push for further treatment in order to stall death™s journey to theirdoorsœmy grandmother lived past years old so ithought my mother would live till atleast without any problems i felt really shocked becausei always thought œshe still has more than years istill have time ¦ so we felt that if it was possibleshe should extend her life dph14 childthe wellbeing determinant n family caregivers reflected on the discovery of positivetheir family member™schanges amidstillness one such change was found within strengthenedkinshipthe trials ofœas we grow up it™s a bit harder [to have familygatherings] because we are all working so when thedisease came even though it™s not a good thing not 0ctanho bmc palliative care page of something you will ask for it united us again maybewithout it [we] would have been a bit more separated ttsh61 childœi feel like we are more united now maybe in thepast we didn't really chat with each other¦ theamount of communication we had increased i feelthat our unity has become stronger dph14 childthey expressed pride in overcoming initial fears by taking charge of and learning to perform unfamiliar taskswith a newfound confidence in their abilities to faceboth practical and emotional difficulties family caregivers also demonstrated a sense of selfempowermentand strengthbased reflection in their sharingœi know nothing about going to visit the government¦ or to do this do that but somehow i findmy way there [i am a] much stronger person so ifanything happens to me i think i know i can faceup to it dph19 spouseœthis is how you grow i learnt to grow because of[my husband] you have to face the insurmountablechallenges that come your way i learnt how toshoulder my responsibilities on my own scs18spousediscussionthis is the first known study that investigates and bringsattention to the internalized motivations of eol familycaregivers while the family dignity intervention studyquestions did not specifically query family caregivers ontheir motives these motivationcentred responses occurred spontaneously and abundantly throughout the interviews “ an indication that internalized motivationsare profoundly espoused within eol caregiving attitudesand behaviors the bobec model fig illustrates theduality ofthese caregiving motivations in regard tomeaningfocused coping and intrapsychic strains as well as identifies an important influence on caregiver wellbeingmotivations with cultural influencessome themes revealed cultural undertones that reflectedthe internalization of asian values into family caregivermotivations in their motivation for alleviating sufferingfamily caregivers displayed the desire to do so by practical means such as administering medication to theirfamily member and experiencing distress when suchmethods were not feasible this is in line with the asianculture of preferring to show concern for their familymembers through pragmatic ways in their motivation for preserving gratitude family caregivers demonstrated the significance of filial piety as well as culturalbeliefs about karma and pastlife within theirattitudes towards caregiving finally in their motivationfor reconciling with mortality family caregivers indicated the importance placed on close intergenerationalconnections as well as longevity for their elders motivations as blessings meaningfocused copinghonoring fidelityall eol caregiving motivationsalleviating suffering enduring attachment preservinggratitude navigating change and reconciling with mortality were found to embody the tenets of meaningfocused coping fuelled by these motivationsfamilycaregivers displayed the propensity for benefitfindingand benefitreminding even in witnessing their familymember™s suffering and imminent mortality adaptivegoal processes in adjusting their expectations and aspirations in accordance with their family member™s physicalcondition and prognosis reordering priorities in hopesof making the most of the time left with their familymember and infusing ordinary events both past andpresent with positive meaning that allowed them to feelaffirmed encouraged and grateful in their daily caregiving as such the capacity for imbuing stressful caregiving events with positive meaning and responseswould make these motivations advocates of perceivedœblessings in the eol caregiving journeymotivations as burdens intrapsychic strainsparadoxically the authors found that these eol caregiving motivations also ran parallel to the intrapsychicstrains as postulated by pearlin and colleagues intheir seminal stress process model intrapsychic strainsoccur when the caregiver™s selfconcept is diminisheddue to the chronicity of providing care intrapsychicstrains unique to caregivers were defined as role captivity in which the caregiver feels entrapped within hisor her role whether or not by personal choice the lossof self in which the caregiver experiences a loss of identity and sense of personhood as enmeshment with thepatient ensues perceived low competencein whichthe caregiver does not see the value and skill of the workthey do leading to a sense of helplessness and perceived lack of gain in which the caregiver does not findpersonal growth orcaregivingprocessenrichmentin theshould their eol caregiving motivations personifythese strains it is only a matter of time before familycaregivers experience outcomes of mental and emotionaldistress such as depression anxiety and irritability aswell as a decline in physical health and a disengagementfrom their caregiving roles in short these motivations would most certainly bludgeon the family caregiverwith great ˜burdens™ within the eol caregiving journey 0ctanho bmc palliative care page of the crucial factor selfdeterminationselfdetermination theory suggests that people needto feel a sense of competence gaining mastery of tasksand having selfefficacy relatedness feeling like theybelong and mutually relating to others and autonomycontrol over their own choices behaviors and goals inorder to fuel high quality motivation that helps one tothrive the theme of the wellbeing determinant alignswith this concept in a caregivingcentric phenomenonfamily caregivers contributing to this theme displayedconfidence in carrying out previously unfamiliar caregiving tasks competence affirmed a stronger sense of kinship relatedness with the patient and their families andtook ownership of their caregiving responsibilities andchallenges autonomy encouragingly numerous studieshave proposed that high quality motivations stemmingfrom selfdetermination can elicit outcomes of greaterfortitude higher commitment and more positive emotions and selfconcepts [“]building on said studies the authors propose that family caregivers who feel a sense of competence relatedness and autonomy within their caregiving motivationswould be further inclined to meaningfocused copingsuch as deriving perceived benefits from their caregiving even in difficult events reminding themselves ofthese benefits when faced with similar circumstances setting their own goals in caregiving having the competence and confidence to be flexible and adaptive and finding positivity in normal everyday situations possessing a sense of selfdetermination in the caregivingrole would in essence safeguard one™s caregiving motivations from the intrapsychic strains of perceived entrapment a sense of disempowermentineptitude andfruitlessnessas such the bobec modelidentifies the wellbeingdeterminant as an indicative element of caregiver selfdetermination and a crucial factor as to whether the eolfamily caregiver perceives their journey as one lined withblessings or laden with burdens competencetargeted mediators apart fromgeneral psychoeducation on symptom managementmedical care and selfcare interventions could incorporate mediums to develop and improve selfefficacy these can take the form of personalstrengths journaling facilitating peer support between new and experienced caregivers rolemodelling and goalsetting such interventions canbe created in the form of structured support groupsonline platforms or mobile applications autonomytargeted mediators in addition toproviding adequate and appropriate eol caregivereducation autonomytargeted interventions suchas those involving mindfulness practice and thearts can serve to give caregivers a sense of control as well as help them make meaning out oftheir thoughts emotions and circumstances todate the mindfulcompassion art therapymcat for eol care professionals showsgreat potential to be converted into a programfor eol family caregivers relatednesstargeted mediators dyadic or familyprojects that recall shared memories expressappreciation seek fiveness impart wisdom andcreate generativity such as the fdi are valuable incrafting the bond of relatedness among familycaregivers and their family members such projectsshould be implemented as a foundation ofpsychosocial interventions at the endoflifeinterventions should be offered to family caregiversin a culturallyrelatable manner this can be donethrough emphasizing how these mediators will helpfamily caregivers enhance their practical caregivingwith increased competencesense of control andmeaningmaking as well as enrich their intergenerational familial bonds with conversations that focus onlegacy creationimplications and recommendationsfindings from a number of studies show that fulfillingone™s sense of selfdetermination appears central to sustaining one™s motivation and innate satisfaction in caregiving [“] the findings from this paper indicatethat caregivers are driven by motivations that couldequally contribute to wellbeing nurturance or diminishment at the same time our findings also indicate thatcaregivers possess a caregivercentric sense of selfdetermination the wellbeing determinant that is theorized to have positive affect on their motivations thusthis paper recommends that caregiver support interventions should comprise all of the following mediators inorder to fulfil the need for selfdeterminationlimitations and future directionswhile the anically occurring responses emphasize thesignificance of motivations within eol caregiving theseresponses were not examined further during the fdi interviews due to other pri

expanding cancer predisposition genes with ultra‘rare cancer‘exclusive human variationsRoni Rasnic1 nathan Linial1 Michal Linial2It is estimated that up to of cancer incidents are attributed to inherited genetic alterations Despite extensive research there are still gaps in our understanding of genetic predisposition to cancer It was theorized that ultrarare variants partially account for the missing heritable component We harness the UK BioBank dataset of individuals of which were diagnosed with cancer to detect ultrarare possibly highpenetrance cancer predisposition variants We report on cancerexclusive ultrarare variations and nominate variants with additional independent evidence as cancer predisposition variants We conclude that population cohorts are valuable source for expanding the collection of novel cancer predisposition genesDiscovery of cancer predisposition genes CPGs has the potential to impact personalized diagnosis and advance genetic consulting Genetic analysis of family members with high occurrences of cancer has led to the identification of variants that increase the risk of developing cancer1 In addition to familybased studies efforts to identify CPGs focus on pediatric patients where the contribution of environmental factors is expected to be small Forty percent of pediatric cancer patients belong to families with a history of cancer2Tumorigenesis results from misregulation of a0one or more of the major cancer hallmarks3 Therefore it is anticipated that CPGs overlap with genes that are often mutated in cancerous tissues Indeed CPGs most prevalent in children TP53 APC BRCA2 NF1 PMS2 RB1 and RUNX12 are known cancer driver genes that function as tumor suppressors oncogenes or have a role in maintaining DNA stability4 Many of the predisposed cancer genes are associated with pathways of DNArepair and homologous recombination5 The inherited defects in cells™ ability to repair and cope with DNA damage are considered as major factors in predisposition to breast and colorectal cancers6Complementary approaches for seeking CPGs are largescale genomeexome wide association studies GWAS which are conducted solely based on statistical considerations without prior knowledge on cancer promoting genes7 Identifying CPGs from GWAS is a challenge for the following reasons limited contribution of genetic heritability in certain cancer types low effect sizerisk associated with each individual variant lowpenetrance in view of individual™s background8 and low statistical power Large cohorts of breast cancer show that of cancer cases are associated with mutations in BRCA1 and BRCA2 which are also highrisk ovarian cancer susceptibility genes Additionally TP53 and PTEN are associated with earlyonset and highrisk familial breast cancer Mutations in ATM and HRAS1 mildly increase the risk for breast cancer but strongly increase the risk for other cancer types and a collection of DNA mismatch repair genes MLH1 MSH2 MSH6 PMS2 are associated with high risk of developing cancer9 A large cohort of Caucasian patients with pancreatic cancer reveal high risk CPGs that overlap with other cancer types CDKN2A TP53 MLH1 BRCA2 ATM and BRCA110Estimates for the heritable component of predisposition to cancer were extracted from GWAS familybased and twin studies11“ These estimates vary greatly with maximal genetic contribution associated with thyroid and endocrine gland cancers and a minimal one with stomach cancer and leukemia14 Current estimates suggest that as many as of cancer incidents can be attributed to inherited genetic alterations eg single variants and structural variations1516 The actual contribution of CPGs varies according to gender age of onset cancer types and ethnicity17“ It is evident that high risk variants with large effect sizes are very rare21 Actually based on the heritability as reflected in GWAS catalog it was estimated that only a fraction of existing CPGs is presently 1The Rachel and Selim Benin School of Computer Science and Engineering The Hebrew University of Jerusalem Jerusalem Israel 2Department of Biological Chemistry Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem Israel email ronirasnicmailhujiacilScientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 UK Biobank CUVs collection The Caucasian filtered UK Biobank UKBB data set include individuals who had cancer and the nonCaucasian include such individuals a Cancer type distribution for the Caucasian data set b Cancer type distribution for the nonCaucasian data set c The data of UKBB participants was used for this study of which were confirmed Caucasian d Out of UKBB variants we curated heterozygous and homozygous CUVs total CUVs known22 Therefore instances of extremely rare mutations with high risk for developing cancer remain to be discoveredA catalog of CPGs was compiled from a0years of research1 with about half of the reported genes derived from family studies representing highpenetrance variants An extended catalog was reported with a total of CPGs that were tested against rare variants from TCGA germline data covering cancer patients from cancer types and included known pediatric CPGs23 The contribution of BRCA12 ATM TP53 and PALB2 to cancer predisposition was confirmedIn this study we report on known and novel cancer predisposition candidate genes We benefit from the UKBiobank UKBB an invaluable resource of germline genotyping data for individuals The UKBB reports on cancer patients and cancer free individuals considered as control group We challenge the possibility that CPGs can be identified from very rare events henceforth called cancerexclusive ultrarare variants CUVs These CUVs are expected to exhibit high penetrance Notably the presented CUVs were extracted from UKBB DNA array and therefore only cover the array preselected SNPs We report on exome variations of which are heterologous The majority of the matching genes are novel CPG a0candidates We provide indirect genomic support for some of the CUVs that occur within coding genes and discuss their contribution to tumorigenesisResultsThe primary UKBB data set used in the is comprised of Caucasian UKBB participants see Methods Fig a01c cancerfree and diagnosed with at least one malignant neoplasm Among participants with cancer were diagnosed with either skin or breast cancer The clinical ICD10 codes assembly is summarized in Supplementary Table a0S1 A total of of the cancerdiagnosed individuals had two or more distinct neoplasms diagnosed The validation UKBB data set includes nonCaucasian participants among them are cancerfree Figure a01ab provide further details on different cancer type prevalence in these setsNonmelanoma skin cancer is mostly attributed to environmental factors rather than genetic association24 However based on evidence for hereditary links for nonmelanoma skin cancer predisposition2526 we included these individuals in our analysis In addition focusing on extremely rare variations enables the identification of existing yet overlooked genetic associationsCompilation of cancerexclusive ultrarare variants CUVs We scanned genetic markers in our prime data set for cancerexclusive variations variations met our initial criteria appearing at least twice in individuals diagnosed with cancer and not appearing in cancerfree individuals Among them were heterozygous and were homozygous variations In order to target variations with additional supporting eviScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Exomic CUVs are mostly gene disruptive The partition of variant types for the compiled list of exomic CUVs The list is dominated by transcript disruptive variations that include missense frameshift stop gain and splicing sites a Distribution of variation types among the exomic CUVs b Dispersion of variant types among heterozygous and homozygous CUVsdence we considered only coding exome and spliceregion variants To assure the CUVs rarity in the general population we applied an additional filter based on the gnomAD data set see Methods The resulting final list is comprised of variants associated with genes heterozygous and homozygous Fig a01d The detailed list of all CUVs can be found in Supplementary Table a0S2Most of the CUVs are missense variants There is a strong enrichment for loss of function LoF variants ie frameshift splicing disruption and stop gains which account for of the CUVs Only a single homozygous CUV is synonymous Fig a02a The distribution of variation types varies greatly between homozygous and heterozygous CUVs Fig a02b Missense variants are of the homozygous variant set but only of the heterozygous CUVs The heterozygous CUVs are highly enriched for LoF variants which constitute the other Cancerexclusive ultrarare variants overlap with known cancer predisposition genes From the listed CUVs variants were previously defined as cancer inducing genes in genes Table a0 Specifically CUVs within genes appear in the updated list of CPG catalog23 and CUVs within genes are known cancer driver genes Fig a03a as determined by either COSMIC27 or the consensus gene catalog of driver genes listing genes coined C29928 More than half of the cancer associated variants result in LoF Many of the affected genes are tumor suppressor genes TSGs among which are prominent TSGs such as APC BRCA1 and BRCA2 Table a0 each identified by two distinct CUVs Notably of the variants had at least one appearance in nonmelanoma skin cancerThe heterozygous CUVs are enriched for known cancer predisposition genes Twentyfive of the cancer associated CUVs are heterozygous and one is homozygous However there is an inherent imbalance in the initial variant sampling performed by the UKBB As the UKBB use DNA arrays for obtaining genomic data the identifiability of ultrarare exome variants is restricted by the selection of SNP markers and the design of the DNA array There are heterozygous ultrarare exome variants from genes which pass our biobankethnic and the gnomAD allele frequency filtration A total of of the filtered ultrarare variants overlap with known CPGs as some genes are overrepresented among the ultrarare variants Supplemental Table a0S3 For example the exomic region of BRCA2 is covered by such SNP marker variants while most genes have noneIn order to account for the disproportional number of the ultrarare variant of some CPGs we calculated the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the collection of heterozygous ultrarare variants As shown in Fig a03b there is an enrichment towards CPGs and even more so as we remove variants of overrepresented genes eg BRCA2 The statistical significance estimates pvalues for each datapoint are available in Supplemental Table a0S3 see MethodsIndependent genetic validation Due to the extremely rare nature of the CUVs we require additional support for the collection of the CPG candidates We seek independent genetic validation of the noncancer related CUVs We apply three sources for validation the filtered Caucasian UKBB cohort the matched filtered nonCaucasian UKBB cohort the collection of germline variants from TCGA as reported in gnomAD The complete list of genetically validated novel CPG candidates is listed in Table a0 Ten out of the novel CPGs were identified based on appearances in individuals with nonmelanoma skin cancerWithin the Caucasian cohort we consider the following as additional genomic evidence a gene with CUVs or any CUV seen in more than two individuals diagnosed with cancer We found genes that have distinct CUVs of which are already known CPGs BRCA1 BRCA2 and APC The other genes are likely novel Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cRefEffecthg19TMissenseGMissenseMissenseTSplice region GSplice region AFrameshiftFrameshiftStop gainMissenseFrameshiftMissenseStop gainMissenseMissense MissenseFrameshiftMissenseFrameshiftFrameshiftMissenseMissenseFrameshiftStop gainFrameshiftSplice region CMissenseTAlt GeneGBACMSH6AVHLGTGFBR2AMLH1GAPCAAGGA APCGTCTGTCC CTG AG TCTTCCGCACAGGCGAACAAGAGCTGGGCCACCGTCTGFBR1SPTAN1RETBMPR1APTENEXT2NUMA1ATMBRCA2BRCA2RB1ERCC5TSC2NF1BRCA1BRCA1TGIF1RUNX1NF2COSMIC C299 CPG FunctionaYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYEnzymeDNA repairUbqcomplexKinaseTSGTSGTSGKinaseCytoskeletalKinaseKinaseTSG PhosphataseTSG EnzymeMT Spindle poleDDR KinaseTSG DNA repairTSG DNA repairTSGDNA repairTSGRAS regulatorTSG DNA repairTSG DNA repairTGF ligandTFCytoskeletalYYYYYYYYYYYYYYYYYYYYTable CUVs overlap with known cancer predisposition or driver genes a Function abbreviation DDR DNA damage response TSG tumor suppressor gene TF transcription factor MT microtubule Ubq ubiquitin Variants with at least one appearance in nonmelanoma skin cancerFigure a0 CUVs list is enriched with cancer predisposition genes Out of the genes in the CUVs list are known cancer genes a Venn diagram of the genes associated with CUVs known cancer driver genes as reported in COSMIC and the consensus CPGs b Expected number of known CPG CUV orange versus the actual number of known CPG in heterozygote CUVs blue An unbalanced representation of genes in ultrarare variants of UKBB results in overrepresentation of some genes We therefore ranked the genes based on number of ultrarare variants Supplementary Table a0S3 For each rank we present the expected number of CUVs from CPGs and the actual number observed for CUVs from CPGsScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cGene SymbolZygote form People per CUV Distinct CUVs NonCaucasian cohortTCGA germlineAGR2AKR1C2DNAH3DSPEGFLAMENDOUHIST1H2BOHSPB2ICAM1ISLRKCNH2MAP3K15MRPL39MYBPC3MYO1ENAV3PCDHB16SARDHSCN5AWDFY4ZFC3H1HeteroHeteroHomoHeteroHeteroHomoHeteroHeteroHomoHomoHeteroHeteroHeteroBothHomoHeteroHomoHomoHeteroHeteroHomoYYYYYYYYYYYYYYYYYFunction in tumorigenesisAffects cell migration transformation and metastasis Wnt signaling tumor antigenExerts an inhibitory effect on oncogenesisCancer predisposed genes in Tunisian familyAffects cell adhesion Suppressed by TGFβPromotes matrix assemblyCancer biomarkerAffects major signaling pathwaysEpigenetically regulatedBiomarker under a clinical trialMarker for mesenchymal stem cells Deregulated gene in cancerAffects proliferation and migrationContributes to cell migrationTumor suppressor by targeting miR130Cytoskeletal modifierStimulates upregulation of motility and invasionActs as a suppressor of breast cancerActs as tumor suppressorPromotes breast cancer possess antipancreatic cancerPresentats viral tumor antigen on dendritic cellsIndirect activating DNA repairRefTable Novel validated CPG candidates Variants with at least one appearance in nonmelanoma skin cancerCPG candidates DSP KCNH2 MYBPC3 and SCN5A There are CUVs which we detected in three individuals with cancer Three of them are known predisposition or driver genes NF1 ATM and TGFBR2 The other genes are CPG candidates that were not previously assigned as such This set includes PCDHB16 DNAH3 ENDOU AGR2 HIST1H2BO and NAV3 Interestingly a certain homozygous CUV in the gene ICAM1 appeared in individuals with cancer in our filtered Caucasian cohortThe nonCaucasian UKBB cohort provides additional independent genomic evidence There are CUVs that appear at least once in an individual with cancer from the nonCaucasian cohort CUVs from the genes MYO1E SARDH and ISLR appeared in two distinct individuals with cancer from this nonCaucasian cohort while CUVs from PCDHB16 and known CPG BMPR1A appeared in a single individual with cancerTCGA germline variants were obtained using exome sequencing and thus offer an additional separate source for CUV validation Clearly the appearance of CUVs in TCGA germline data is not anticipated as we discuss variants that are ultrarare in both UKBB and gnomAD The TCGA collection within gnomAD includes only samples We identified CUVs that were also observed in TCGA gnomAD germline data one of a known cancer driver gene TGIF1 and novel CPG candidates PCDHB16 EGFLAM AKR1C2 MAP3K15 MRPL39 DNAH3 WDFY4 HSPB2 and ZFC3H1Based on the above support we compiled a list of validated CPGs which includes genes that are novel CPGs Among these genes CUVs are heterozygous are homozygous and MYBPC3 is supported by both heterozygous and homozygous CUVs Two of these genes have multiple validation evidence DNAH3 with a homozygous CUV which appears in individuals with cancer in the Caucasian cohort and within TCGA germline variant collection PCDHB16 with a homozygous CUV which appeared in individuals in the Caucasian cohort one individual in the nonCaucasian cohort and in the TCGA gnomAD resource In addition nonCPG cancerdriver genes with validated CUVs include TGFBR2 and TGIF1 that are also very likely CPG candidatesSome of the prominent genes in our list were signified by additional independent studies For example a novel oncolytic agent targeting ICAM1 against bladder cancer is now in phase of a clinical trial29 Additionally DNAH3 was identified as novel predisposition gene using exome sequencing in a Tunisian family with multiple nonBRCA breast cancer instances30Somatic mutations in novel CPGs significantly decrease survival rate There is substantial overlap between CPGs and known cancer driver genes Fig a03a This overlap suggests that somatic mutations in validated CPG candidates may have an impact on patients™ survival rate We tested this hypothesis for the novel CPG candidates Table a0 using a curated set of nonredundant TCGA studies compiled in cBioPortal3132 that cover patients By testing the impact of alteration in the novel CPGs in somatic data we expect to provide a functional link between the germline CPG findings and the matched mutated genes in somatic cancer samples Altogether of the patients had somatic mutations in one or more of the genes The median survival of patients with somatic mutations in these genes is a0months while the median for patients without Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Somatic mutations in CPG candidate effect cancer patient survival and disease progression The effect of somatic mutations in the novel CPG candidate Table a0 on the survival rate of TCGA cancer patients was tested via cBioPortal a Meier“Kaplan survival rate estimate b Meier“Kaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the Kaplan“Meier survival estimate yields a p value of 178eˆ’ in the Logrank test Fig a04a The Kaplan“Meier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603eˆ’ Fig a04b Cancer types in this analysis are represented by varied number of patients and percentage of individuals with somatic mutations in any of the novel CPGs Supplemental Table a0S4 The trend in most cancer types match the presented pancancer analysis Survival and diseaseprogression estimate for each cancer type are available in Supplementary Figures a0S1“S24 Hazard Ratios and confidence intervals were calculated see Materials and Methods and Supplemental Table a0S4We conclude that the CUVbased CPG candidate genes from UKBB carry a strong signature that is manifested in patients™ survival supporting the notion that these genes belong to an extended set of previously overlooked CPGsHomozygous variations are mainly recessive In order to ascertain whether the homozygous variations found are indicative of the heterozygous form of the variant as well we viewed the heterozygous prevalence within the UKBB Caucasian population In only a single variant in the gene MYO1E was the prevalence in healthy individuals significantly lower than in individuals with cancer p value As most of the variations have a strong cancer predisposition effect as homozygous variations it seems that their influence is explained by a recessive inheritance mode This phenomenon might explain the significant depletion of known CPGs within the homozygous variations in our listInspecting the heritability model of previously reported CPGs1 is in accord with our findings showing that while about twothirds of the genes comply with a dominant inheritance the rest are likely to be recessive Notably in the most updated CPG catalog of the genes were assigned with both inheritance patterns In our ultrarare list only MYBPC3 is associated with both heterozygous and homozygous variationsDiscussionWe present a list of CUVs from genes Among them variants from genes are associated with known cancer genes Most of these variants overlap with known cancer predisposition genes Expanding the number of currently identified CPGs is crucial for better understanding of tumorigenesis and identifying various processes causing high cancer penetrance Genetic consulting family planning and appropriate treatment is a direct outcome of an accurate and exhaustive list of CPGsKnown cancer predisposition variants only partially explain the cases of inherited cancer incidents CPGs identification has already impacted cancer diagnostics therapy and prognosis1 Genomic tests and gene panel for certain cancer predisposition markers are commonly used for early detection and in preventative medicine3334 It is likely that CPGs based on ultrarare variants are not saturated For example additional CPGs including CDKN2A and NF1 were associated with an increased risk for breast cancer35 Specifically CDKN2A has been also detected as a CPG in families of patients with pancreatic cancer36 Inspecting the function of genes associated with Scientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cthe identified genes further supports the importance of protein modification eg kinases and phosphatase function chromatin epigenetic signatures37 membrane signaling DNA repair systems and moreNumerous CUVs are present in individuals with nonmelanoma skin cancer For the most part nonmelanoma skin cancers are attributed to environmental factors Nevertheless studies show that there are in fact genetic components associated with the majority of nonmelanoma skin cancers2526 Accordingly CUVs can unveil such rare genetic associationsWe chose to focus on cancerexclusive variants to shed light on mostly overlooked ultrarare cancer predisposition variants Naturally additional ultrarare variants in the dataset are presumably cancer inducing Detecting these variants requires developing a broader model expanding the scope to somewhat less rare possibly lowerpenetrance variants The impending availability of UKBB exome sequencing exomes will enable us to revisit the identified variants to further refine the list of candidate CPGs ie removing falsepositives and adding evidence to support true CPGs and to develop a less strict detection modelThe inheritably rare nature of CUVs raise concerns on the reliability of their initial identification38 We overcome this hurdle by only considering as candidate CPGs those genes that are supported by additional independent genomic evidence from either the UKBB or the TCGA cohort We nominate genes as CPG candidates two of which are known cancer drivers As we have shown Fig a0 somatic mutations in the nondriver validated CPG candidates resulted in a significant negative effect on the patients™ survival rateMaterials and methodsStudy population The UKBB has recruited people from the general population of the UK using National Health Service patient registers with no exclusion criteria39 Participants were between and a0years of age at the time of recruitment between and To avoid biases due to familial relationships we removed samples keeping only one representative of each kinship group of related individuals We derived the kinship group from the familial information provided by the UKBB fam files Additionally samples had mismatching sex between the selfreported and the geneticsderived and samples had only partial genotypingWe divided the remaining participants into two groups ˜Caucasians™”individuals that were both genetically verified as Caucasians and declared themselves as ˜white™ ˜nonCaucasians™”individuals not matching the previous criterion The Caucasian cohort includes individuals of whom had cancer and the nonCaucasian cohort includes individuals had cancer We used the Caucasian cohort for our primary analysis and the nonCaucasian cohort for additional validation purposesVariant filtration pipeline We considered a heterozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation and no healthy individuals with the variation in the Caucasian cohort We considered a homozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation ie homozygous to the alternative SNP and no healthy individuals with the homozygous variation in the Caucasian cohort The ensemble Variant effect predictor40 was used to annotate the variantsWe applied two additional filtration steps for the exomesplicingregion variants The first filter was applied using the ˜nonCaucasian™ data set we filtered heterozygous variations with MAF and homozygous variations with homozygous frequency in this set This filtration step is meant to diminish variations which are mostly ethnic artifacts The second filter was applied to assure the variations rarity We applied the same filter heterozygous variations with MAF and homozygous variations with homozygous frequency using gnomAD v21141 The used gnomAD threshold was based on the summation of gnomAD v211 exomes and genomes We also used gnomAD for the TCGAgermline validation by extracting TCGA appearances from the databaseStatistical analysis The UKBB ultrarare variants are enriched with CPGs variants We accounted for this imbalance by calculating the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the ultrarare variant collection for heterozygotes We calculated pvalues for each datapoint using a twoside binomial testWe downloaded survival data from cBioPortal The data only included survival months We used Cox regression without covariates to calculate Hazard Ratio and confidence intervals The results are listed in Supplementary Table a0S4Rare variants reliability Our CUV collection includes variants that appeared at least twice in the filtered Caucasian cohort thereby evading many SNPgenotyping inaccuracies38 We further ascertain the validity of prominent variants with additional genomic evidenceCancer type definition The UKBB provides an ICD10 code for each diagnosed condition We considered an individual diagnosed with malignant neoplasm ICD10 codes C00C97 as individuals with cancer and otherwise as cancerfree individuals The codes were aggregated to improve data readability using the assembly described in Supplementary Table a0S1Ethical approval All methods were performed in accordance with the relevant guidelines and regulations UKBB approval was obtained as part of the project Ethical approval for this study was obtained from the Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0ccommittee for ethics in research involving human subjects for the faculty of medicine The Hebrew University Jerusalem Israel Approval Number UKBB received ethical approval from the NHS National Research Ethics Service North West 11NW0382 UKBB participants provided informed consent forms upon recruitmentData availabilityMost of the data that support the findings of this study are available from the UKBB However restrictions apply to the availability of these data which were used under license for the current study and so are not publicly available Data are available from the authors upon a justified request and with permission of the UKBB Data extracted from gnomAD is available from the authors upon requestReceived February Accepted July a1508 References Rahman N Realizing the promise of cancer predisposition genes Nature 101038natur e1298 Zhang J et al Germline mutations in predisposition genes in pediatric cancer N Engl J Med 101056NEJMo Hanahan D Weinberg R A Hallmarks of cancer the next generation Cell 101016jcell201102013 Vogelstein B Kinzler K W Cancer genes and the pathways they control Nat Med 101038nm108 Bertelsen B et al High frequency of pathogenic germline variants within homologous recombination repair in patients with advanced cancer npj Genom Med 101038s4152 Easton D F How many more breast cancer predisposition genes are there Breast Cancer Res 101186bcr6 Hindorff L A et al Potential etiologic and functional implications of genomewide association loci for human diseases and traits Proc Natl Acad Sci U S A 101073pnas09031 Galvan A Ioannidis J P A Dragani T A Beyond genomewide association studies genetic heterogeneity and individual predisposition to cancer Trends Genet 101016jtig200912008 Baria K Warren C Roberts S A West C M Scott D Chromosomal radiosensitivity as a marker of predisposition to common cancers Br J Cancer 101054bjoc20001701 Hu C et al Association between inherited germline mutations in cancer predisposition genes and risk of pancreatic cancer J Am Med Assoc 101001jama20186228 Verkasalo P K Kaprio J Koskenvuo M Pukkala E Genetic predisposition environment and cancer incidence a nationwide twin study in Finland “ Int J Cancer 101002SICI1097021519991 210836743AIDIJC830CO2Q Frank S A Genetic predisposition to cancer”insights from population genetics Nat Rev Genet 101038nrg14 Law P J et al Association analyses identify new risk loci for colorectal cancer susceptibility Nat Commun 101038s4146 w Czene K Lichtenstein P Hemminki K Environmental and heritable causes of cancer among million individuals in the Swedish FamilyCancer Database Int J Cancer 101002ijc10332 Economopoulou P Dimitriadis G Psyrri A Beyond BRCA new hereditary breast cancer susceptibility genes Cancer Treat Grant R C et al Prevalence of germline mutations in cancer predisposition genes in patients with pancreatic cancer GastroenRev 101016jctrv201410008 terology 101053jgastr o201411042 Petersen G M et al A genomewide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q221 1q321 and 5p1533 Nat Genet 101038ng522 Wolpin B M et al Genomewide association study identifies multiple susceptibility loci for pancreatic cancer Nat Genet Long J et al Genomewide association study in East Asians identifies novel susceptibility loci for breast cancer PLoS Genet 101038ng3052 101371journ alpgen10025 Thomas G et al Multiple loci identified in a genomewide association study of prostate cancer Nat Genet 101038ng91 Mancuso N et al The contribution of rare variation to prostate cancer heritability Nat Genet 101038ng3446 Jiao S et al Estimating the heritability of colorectal cancer Hum Mol Genet 101093hmgddu08 Huang KL et al Pathogenic germline variants in adult cancers Cell 101016jcell201803039 Griffin L L Ali F R Lear J T Nonmelanoma skin cancer Clin Med J R Coll Physicians Lond 107861 Nikolaou V Stratigos A J Tsao H Hereditary nonmelanoma skin cancer Semin Cutan Med Surg 101016jclinm edici ne16162 sder201208005 Roberts M R Asgari M M Toland A E Genomewide association studies and polygenic risk scores for skin cancer clinically useful yet Br J Dermatol 101111bjd17917 Forbes S A et al COSMIC exploring the world™s knowledge of somatic mutations in human cancer Nucleic Acids Res 101093nargku10 cell201802060 Bailey M H et al Comprehensive characterization of cancer driver genes and mutations Cell 101016j Annels N E et al Phase I trial of an ICAM1targeted immunotherapeuticcoxsackievirus A21 CVA21 as an oncolytic agent against non muscleinvasive bladder cancer Clin Cancer Res 10115810780432CCR184022 Hamdi Y et al Family specific ge

American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ‰¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was “ mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research “ Fu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the work you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ‰¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundary“lower hyoid margin lower boundary“superior sternal fossa upper margin of the unknown artery and external“lateral margin of the carotid sheath and lower boundary“anterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ‰¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients™ complete medical records were available All of the surgeons had years™ experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the œat least  principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using χ2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients™ age range was “ years old with a median of ± years The maximum diameter range of the cancer lesion was “ mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ‰¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients™ gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimoto™s thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimoto™s thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1“ According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about “ On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8“ These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japan™s Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ‰¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ‰¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 œ American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimoto™s thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter‰¤ mm mm x ‰¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMχ2PYes n No n “““Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβˆ’ˆ’SEWaldPOR CI OR““““““““Abbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ‰¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The χ2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions χ2 P nodule morphology χ2 P and extra thyroidal expansion of cancer focus χ2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimoto™s thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol “ doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg “ doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence “ Cancer Causes Control “ doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid “ doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA “ United States “ doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid “ doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery “ discussion “ doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin “ doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery “ doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid “ doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid “ doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary “ doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol “ doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid “ doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res “ doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid “ doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c'

"Despite recent interest in the use of ketogenic diets KDs for cancer evidence of beneficial effects islacking This study examined the impact of a randomly assigned KD on quality of life physical activity andbiomarkers in patients with breast cancerMethod A total of patients with locally advanced or metastatic breast cancer and without a history of renaldisease or diabetes were randomly assigned to either a KD or a control group for this 12week trial Concurrentwith the first third and fifth chemotherapy sessions quality of life physical activity and biomarkers thyroidfunction tests electrolytes albumin ammonia ALP lactate and serum ketones were assessed Dietary intake wasalso recorded on admission and the end of the treatmentResults No significant differences were seen in quality of life or physical activity scores between the twogroups after weeks however the KD group showed higher global quality of life and physical activityscores compared to the control group at weeks P P Also serum lactate and ALP levelsdecreased significantly in the KD group compared to the control group at the end of the intervention ± vs ± ± vs ± P and P respectively A significant inverse associationwas observed between total carbohydrate intake and serum betahydroxybutyrate at weeks r ˆ’ P No significant differences between groups were observed in thyroid hormones electrolytes albuminLDH or ammonia Compliance among KD subjects ranged from to as assessed by dietary intakeand serum ketones levels of Continued on next page Correspondence khodabakhshiadelehyahoocom6Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKhodabakhshi Nutrition Journal Page of Continued from previous pageConclusion According to our results besides a higher global quality of life and physical activity scores compared tothe control group at weeks KD diet combined to chemotherapy in patients with breast cancer does not bringadditional benefit about quality of life and physical activity at weeks However decreases seen in levels of lactateand ALP in the KD group suggest that a KD may benefit patients with breast cancerTrial registration This trial has been registered on Iranian Registry of Clinical Trials IRCT under the identification codeIRCT20171105037259N2 wwwirctirtrial30755Keywords Ketogenic diet Breast cancer quality of life Physical activity Lactate Alkaline phosphatase chemotherapythere are stillIntroductionKetogenic diets KDs are high in fat and very low incarbohydrate They have been used as a dietary treatment in epilepsy for nearly a century [] RecentlyKDs have gained the attention of cancer researchersdue to their potential impact on cancer cell metabolism [] Despite the growing evidence of possibleantitumor benefitssome concernsabout potential adverse effects of KDs in cancer patientsincluding micronutrient deficiencies appetitereduction nausea constipation [] fatigue [] hyperlipidemia and especially unintended weight loss [ ]KDs are perceived as restrictive in nature which mayadd to the burden of cancer patients who already suffer from considerable physical emotional and financialstress all of which are known to negativelyimpact quality of life QoL In addition alterations inphysical and cognitive function during cancer treatment are pervasive It is estimated that “ of patients undergoingfromcancerrelated fatigue [] Prior studies have foundthat KD may improve physical and mental wellbeing[] Less fatigue has been reported in healthy overweight and obese adults following lowglycemic compared to highglycemic diets [] Results ofthreestudies using the validated European anization forResearch and Treatment core QoL questionnaire tofindings [“] Aassess fatigue lacked consistentin advanced cancer patients showed imsmall trialprovementin sleep and emotionalfunction after athreemonth KD intervention [] Other studies havesuggested enhanced cognitive function [ ]cancertreatmentsufferTo date only four studies have assessed QoL in adultpatients with cancer [ “] Hunger is a reported sideeffect of restricted KDs however previous research hasfound that perceived hunger is reduced in low carbohydrate diets compared to low fat diets [] A recent systematic review has highlighted the need for additionallarger investigations on the impact of ketogenic diets onQoL [] The goal of this present trial was to assesswhether a KD had beneficial effects on QoL dietary intake physical activity and specific biomarkersinindividuals with breast cancer while also evaluating compliance to KD guidelines in these patientsThe protocol used in this trial [] and part of the resultsfrom this trial have been previously published [ ]MethodsThe study protocol was approved by the National Nutrition and Food Technology Research InstituteNNFTRI Shahid Beheshti University of MedicalSciencesIRSBMUNNFTRIREC1396187 All participants provided writteninformed consent prior to participating in the studySBMU TehranIranThis trial was a randomized controlled labelclinical trial to breast cancer patients with locally advanced or metastatic disease who were receiving chemotherapy for atleast weeks The studythe medical oncology clinic atwas conducted atShohadaeTajrish hospital Cancer Research CenterTehran Iran from July to October of Participation was to patients to years of ageExclusion criteria screened forsignificant cardiacrenal or neurologic comorbidities symptoms of malnutrition diabetes pregnancy and Karnofsky indexless than Using a block balanced randomizationmethod patients were assigned to the interventionn or controln groups Randomizationwas computergenerated by a statistician who was nota member of the medical team Blinding the participants or study personnel was not deemed feasible inthis dietintervention The project coordinator enrolled the participants and assigned them to their interventions Both the KD and the control diet werecalculated to be eucaloric using the MifflinSt Jeorformula The KD consisted of of calories fromCHO from protein from mediumchain triglyceride MCT oil and from fat A dietitianprovided specific nutritional counseling to each participantfacetoface meetings Patientsengaged in ongoing weekly counseling sessions viaphone WhatsApp or Telegram and were assessed forcompliance and possible adverse effects To furtherenhance compliance dietary recommendations werein individual 0cKhodabakhshi Nutrition Journal Page of individualized and appropriate recipes were providedto patients in the KD group were asked to refrainfrom eating any grains grain products starchy vegetables fruit or sugar Dietary carbohydrates were limited to nonstarchy vegetables and dietary proteinswere obtained primarily from egg meat poultry andfish Small amounts of lower carbohydrate berries andnuts were allowed as long as they did not exceed thecarbohydrate limit in the diet prescription Subjectswere encouraged to increase their fat intake and toselect from a variety of sourcesincluding olive oilbutter and cream cheese Patients were asked tochoose only the foods specified in the diet plan provided to them Patients were also encouraged to usemediumchain triglyceride MCT oil MCT oil anodorless and tasteless saturated fat does not requirebile or pancreatic enzymes for digestion It is easilyconverted to ketones in the liver thereby enhancingketosis Every weeks ml of MCT oilfromNutricia Erlangen Germany was provided to eachsubject in the KD group For better tolerance initialdosage of MCT was kept low and increased daily overa 6day period until maximum tolerable dosage wasachieved Dosage was reduced in a similar steppedprocessThe patients in the control group were instructed tofollow a standard diet consisting of CHO protein and fat Dietary compliance was checked byassessing blood betahydroxybutyrate levels every weeks and dietary intake at baseline and end of thestudyQoL assessmentQoL was assessed using the EORTC QLQC30 version and IORTC QLQBR23 questionnaires developed bythe European anization for Research and Treatmentof Cancer The validity and reliability of the questionnaires has previously been evaluated in Iran [ ]The questionnaires were completed at enrollment at weeks and at the end of the interventionDietary intake assessmentHospital dietitians used a 24h dietary recall 24HR toobtain a total of days intake one weekend day andtwo workdays through telephone and facetoface interviews both at the beginning and end of the study Theamount of each food consumed was estimated usingcommon household containers bowls cups and glassesand standard measuring cups and spoons as referencesThe mean quantity of total energy carbohydrate proteinand fat were estimated from the 24HRDietary intake wasanalyzed by Nutritionist IV software Version USPhysical activity assessmentPhysical activity was measured using the IPAC International Physical Activity questionnaire at baseline at weeks and at the end of the studyBiomarker assessmentFasting blood sampling for serum Na K Ca P lactate Mg LDH albumin ammonia and ALP were performed at baseline midway through the intervention weeks and at weeks T3 T4 and TSH were measuredat baseline and the end of the interventionStatistical analysisConsidering the power and α the sample sizewas calculated as individuals per group Assuming a dropout during the weeks of the study the finalnumber of participants was calculated as patients ineach groupStatistical analysis was carried out according to theintentiontotreat protocol Continuous variables weretested for normal distribution by the KolmogorovSmirnov test and then reported as mean ± standard deviation or median as appropriate Student ttest or Mann“Whitney U test was used to compare the continuousvariables between the two groups Paired sample ttestor Wilcoxon was used to compare the continuous variables within the two groups The ANCOVA test wasused to eliminate the effect of confounding factorsPearson correlation analyses were used to estimate associations between total carbohydrate intake and serumbetahydroxybutyrateData were analyzed using the SPSS version software Chicago IL USA and Stata version P was considered as statistically significantResultsDetailed patient demographics and a flow diagram werereported previously [] A total of women withbreast cancer were enrolled and randomly assigned to either the intervention n or control n groupsThree patients in the control group withdrew before beginning their assigned diet while10 patients in the KDgroup and patients in the control group withdrewfrom the study after beginning their assigned diet Ultimately patients in each group completed the studyand were included in the analysis No significant differences were seen between the two groups with regard toage cancer type metastasis and marriage or educationstatus P The intervention group included patients with locally advanced disease and patients withmetastatic disease liver bone lung liver andbone while the control group consisted of patientswith locally advanced disease and patients with 0cKhodabakhshi Nutrition Journal Page of metastatic disease bone liver lung liver andbone at other sites P Table Data related to quality of life are shown in Tables and No significant differences were seen in QoL betweenthe two groups after weeks however the KD groupshowed better global QoL compared to the controlgroup at week P Also at week diarrhea increased in the control groupcompared to the intervention P Data on week not shown Using the QoL questionnaire there was awithingroup decrease in reported hunger from baselineto weeks in the KD group P A withingroupdecrease was seen in physical performance measuresfrom baseline to weeks in both groups which was significant only in the KD group P In addition rolefunctioning and socialfunctioning scores significantlydecreased in the control group compared to the baselinebut not in the KD group P P Table Mean dietary intake is shown in Table and Fig The mean caloric and carbohydrate intake decreasedsignificantly at the end of the study compared to control P and P respectively while fatintake increased significantly in the KD group compared to the control group P After adjustingfor total energy intake this difference remained significant When data from both groups was combineda significant inverse association was observed betweentotalserum betaandr ˆ’ P hydroxybutyratealthough this effect was not seen when the KD groupwas analyzed separatelyintakeat weekscarbohydrateWithingroup analysis showed significant decreasesin energy carbohydrate and protein intake in bothgroups compared to the baseline Fat intake increasedsignificantly compared to the baseline in the KDgroup P and decreased significantly in thecontrol group P During the intervention of the subjects in the KDarm limited carbohydrates to g and of subjects consumed of calories from carbohydratesAt weeks of patients in the KD group hadserum ketones mmolL at 6weeks had ketone levels of mmolL As previously reportedserum ketone concentrations increased significantly inthe KD group ± to ± mmollP []At weeks physical activity improved in the KD groupcompared to the control group adjusted for cancer typeand baseline value P but after weeks physicalactivity did not show any significant differences in a between or withingroup analysis Fig No significant difference was observed in a betweenor withingroup analysis of thyroid hormones electrolytes albumin Ammonia and LDH Howeverlactateand ALP decreased significantly after intervention in theKD group compared to the control group P andP respectively ALP is adjusted for baseline valueand cancer type Table Data on thyroid hormones notshownDiscussionThe effect of KD on QoL physical activity dietary intake and biomarkers in patients with locally advancedand metastatic breast cancers was evaluated in thisstudy Based on our findings in the KD group globalQoL was higher at weeks perhaps in part because diarrhea was more frequent in the control group than theKD group No significant differences were seen in theQoL physical activity and biomarkers between the twogroups after the week intervention Lactate and ALPwere lower in the KD group compared to the controlEffect of diet on QoLIn our study in the KD group global QoL was higher at weeks No adverse effects were observed in thoseTable Baseline characteristics in breast cancer patients before interventionScale categoriesCancer TypeLocally AdvancedIntervention Ketogenic dietn Control Ordinaryn ERPRHER2Metastaticpositivenegativepositivenegativepositivenegative ER Estrogen receptor PR Progesterone receptorHER2 Human epidermal growth factor receptor aCalculated by chi square testbCategorical data shown as No p value008a057a043a079a 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients™ before andafter intervention in KD group and control group as measuredby the EORTC QLQC30aFunctioningaPhysical functioningMD CIControlKDpvalueTable Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQC30SymptomsaFatiguepvalueControlKD ˆ’Week Week pvalue“33a“Nausea and vomitingWeek Week pvalueRole functioningWeek Week pvalue ± 11a ± ± ± Cognitive functioningWeek Week pvalue ± ± Emotional functioningWeek Week pvalueSocial functioningWeek Week pvalue ± ± ± ± Global quality of lifeWeek Week pvalue ± ± ± ± ± ± ± ± ± ± ± ± ± ± ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’After adjusting for baseline value and chemotherapy status no significantdifferences were observedStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the two groupsData shown as mean and SDaThe higher values indicate higher level of functioning and quality of lifeparticipants assigned to the KD compared to the controlgroup after weeks Withingroup analysis showed decreased hunger and physical function in the KD groupcompared to the baseline In the control group role andsocial functioning decreased significantly compared tobaselineResults of a systematic review and metaanalysis haveshown that KDs suppress appetite [] Decrease in hunger or appetite in our study may be due to the high fatcontent of the KD as it decreases the ghrelin releasewhich in turn may reduce appetite High fat intake alsoslows digestion which could also impact the perceptionof hunger Previously we have shown that the KD resultsin weight loss [] As a clinical benefit KDinduced““ ““““““““““ “ “ “ “ “ “Week Week pvaluePainWeek Week pvalueReduction in appetiteWeek Week pvalueSleep difficultiesWeek Week pvalueDyspneaWeek Week pvalueConstipationWeek Week pvalueDiarrheaWeek Week pvalueFinancial concernsWeek Week pvalue““““““““““ “ “ “ “ “ “Mann“Whitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate a higher grade of symptoms Data shown asmedian and quartile 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQBR23aKDControlpvalue “ “ “ “aFunctioningFuture perspectiveWeek Week pvaluebSymptomsArmWeek Week pvalueBreastWeek Week pvalue “ “ “ “Week Systemic therapy side effects “ “Week pvalueConcerns over hair lossWeek Week pvalue “ “ “ “ “ “ “ “Table Comparison of mean ± SD macronutrient intake atbaseline and 12weeksVariableMD CIpvalueKDMean ± SDControlMean ± SDEnergy KcaldayBefore ± ± ± AfterpvalueCarbohydrate grBefore ± ± ± ± AfterpvalueProtein grBeforeAfterpvalueFat gr ± ± ± ± ± ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ 0001a ˆ’ˆ’ ˆ’041aBefore ± ± ˆ’ˆ’ ± ± AfterpvalueStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the groupsMD Mean differenceCI Confidence intervalaAncova Adjusted for baseline value and energy0001aMann“Whitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate higher level of functioning and quality of lifebThe higher values indicate a higher grade of symptomsData shown as median and quartile decreases in appetite weight and body fat may result infavorable changes in breast cancer patients notably inoverweight or obese women [ ]In contrast with our findings Cohen found that aKD significantly enhanced physical function scores inwomen with ovarian or endometrial cancer comparedto the control group but appetite did not change atthe end of the study compared to the baseline []Part ofstudy andCohen™s trial may be explained by the design of thestudy While only of the participants in the Cohen study were undergoing chemotherapy all of ourpatients were receiving treatmentthe inconsistency between ourAlso timing of the administration of the questionnaires and whether the participants were in positive ornegative energy balance may have influenced ourfindingsNo significant difference was reported in QoL at theend of study compared to the baseline by TanShalaby [] However a slight decrease in physical androle functioning as well as temporary constipation andfatigue were reported in the KD group in one study []In our study constipation was noted by participants inthe KD arm during the early days which was managedby dietary changesAlso after weeks in the KD group physical activityscores was higher compared to the control group but at weeks differencessignificantbetween the two groupsin scores were notDietary intake and adherenceOur study data showed a significant decrease in carbohydrate intake and a significant increase in fat intake inthe KD group compared to the control Protein intakewas not significantly different between the two groupsbut decreased overall in both groups when compared tobaseline Total daily carbohydrate intake was similar toresults in the Cohen study [] We also assessed serumbetahydroxybutyrateIn the KD group ofpatients at weeks and at 6weekshad serum ketones and patients at weeks and weeks 0cKhodabakhshi Nutrition Journal Page of Fig Mean caloric intake and distribution of macronutrients as percentage of total kilocalories before and after week intervention in breastcancer patients in two groupsFig Comparison of trend changes in physical activity in breast cancer patients in two groups 0cKhodabakhshi Nutrition Journal Page of LBsvskwleuavpitnopdmisvskwleuavpLBsviitnopdmeuavlpinorrefnoBnodesabldetauclacerewseuavlpllaerusaemdetaepeRepytlsisyanAlavretnIecnedifnoCICecnereffiDnaeMDMpuwolloftsalroskeewskeewkeewropuwolloftsitnopdMiepytrecnacdnaeuavlenilesabrofdetsudAjavocnAsnosirapmoclepitlumrofnoitcerrocˆ’±±±±ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’skeew±±itnopdMi±±enilesaB±±±±±±smArlairTDKICDMlortnoClortnoCDKlebairaVetatcaLluHDLstneitaprecnactsaerbdetaertDKdnalortnocnislevelrekramoBielbaTˆ’ˆ’ˆ’ICDM±±±±lortnoCDKldgcmianommAˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’ˆ’ˆ’ˆ’ICDM±±±±±±lortnoCDK±±±±±±lortnoCDKˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’±±±±±±±±±±±±ICDMlortnoCDKlortnoCDK±±±±ˆ’±±±±ˆ’±±lortnoCDKICDMˆ’±±ICDMlortnoCDKˆ’ˆ’ˆ’ˆ’ˆ’ICDM±±±±±±lortnoCDKLdgmgMldginmubAllqemKlDgmPPLAldgmaClqemaNenilesaBLB 0cKhodabakhshi Nutrition Journal Page of had serum ketones mmoll Cohen reported that of patients had betahydroxybutyrate concentrations mmollA recent systematic study of KDs in adult cancerpatients reported a range of to with a adherence rate overall reported by [] According toour data the level of adherence to the KD interventionsuggests that the diet is a feasible option for women withbreast cancer who are receiving chemotherapyDespite the lack of any restriction in calorie intakein the study design and consistent with findings ofCohen [] the KD group showed a significant reduction in calorie intake compared to the control groupThe decrease in calorie intake may be due to reductions in appetite associated with ketosis as the subjects in the KD arm did not consume all of the fatcalculated for their diet This may also be due in partto customary practices surrounding meal preparationA decrease in appetite and subsequentinadvertentcalorie restriction most often results in weight loss inthe absence of malnutrition or cachexiathis mayhave antiinflammatory and proapoptotic propertieswhich in turn may exert a positive effect on thesehallmarks of cancer Ketosis may also enhance theeffectiveness of chemotherapy while reducing the sideeffects of treatment [ ]Effect of diet on biomarkersConsistent with the outcomes of the previous studiesour results revealed that the KD had no adverse effecton thyroid hormones electrolytes LDH urea and albumin Significant decreases were seen in serum levels oflactate KDs reduce glycolytic activity which in turn mayslow metastases by reducing the acidity of the tumormicroenvironment and lowering the availability of lactate as a substrate for biomass synthesis [] Decreaseswere also seen in ALP High levels of ALP in breast cancer patients is a negative prognostic marker often indicating progression of metastatic disease [] Moreresearch is needed to assess whether lower ALP and lactate as seen in this study contributes to slower rates ofdisease progressionTo our knowledge this is the first randomized controlled trial examining the effects of a KD on QoL inbreast cancer patientsThe primary limitation of this study was the heterogeneous nature of the sample in regards to cancer stageA secondary limitation was the small sample sizeConclusionAccording to our results besides a higher global QoLand physical activity scores compared to the controlgroup at weeks KD diet combined to chemotherapy inpatients with breast cancer does not bring additionalbenefit about QoL and physical activity at weeksWhile many blood biomarkers did not differ significantlybetween the two groups ketosis may still offer benefit tosome patients with breast cancer in part by decreasinglactate and ALPSupplementary informationSupplementary information accompanies this paper at doi101186s1293702000596yAdditional file figure Flow diagram of the patient treatmentprocessAdditional file figure Median confidence interval tyroidhormones in baseline and 12week by two trial arms in breast cancerpatientsAcknowledgmentsWe would like to thank all the patients at our clinic who took part in thisstudyAuthors™ contributionsKhodabakhshi carried out the conception Methodology performed theexperiments design of the diet and wrote the Davoodi and Seyfriedcollaborated in the design of the study Davoodi supervise on the thesisKalamian and Beheshti collaborated in the design of the diet Kalamian gavecritical review of the manuscript All authors have read and approved thefinal manuscriptFundingNot applicableAvailability of data and materialsData described in the manuscript code book and analytic code will bemade available upon request pendingEthics approval and consent to participateAll participants provided written informed consent prior to participating inthe studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nutrition School of Public Health Kerman University ofMedical Sciences Kerman Iran 2Physiology Research Center KermanUniversity of Medical Sciences Kerman Iran 3Biology Department BostonCollege Chestnut Hill MA USA 4Dietary Therapies LLC Hamilton MT USA5Department of Nutrition and Dietetics Mofid children™s hospital ShahidBeheshti University of Medical Sciences Tehran Iran 6Cancer ResearchCenter Shahid Beheshti University of Medical Sciences Tehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranReceived March Accepted July ReferencesNeal EG Chaffe H Schwartz RH Lawson MS Edwards N Fitzsimmons G The ketogenic diet for the treatment of childhood epilepsy arandomised controlled trial Lancet Neurol “Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab 0cKhodabakhshi Nutrition Journal Page of adverse effects on blood lipids a randomized controlled trial Nutr Cancer“Fine EJ SegalIsaacson CJ Feinman RD Herszkopf S Romano MC TomutaN Targeting insulin inhibition as a metabolic therapy in advancedcancer a pilot safety and feasibility dietary trial in patients Nutrition“ Epub Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab Mukherjee P Mulrooney TJ Marsh J Blair D Chiles TC Seyfried TNDifferential effects of energy stress on AMPK phosphorylation and apoptosisin experimental brain tumor and normal brain Mol Cancer Gatenby RA Gawlinski ET Gmitro AF Kaylor B Gillies RJ Acidmediatedtumor invasion a multidisciplinary study Cancer Res “Singh A Pandey A Tewari M Kumar R Sharma A Singh K Advancedstage of breast cancer hoist alkaline phosphatase activity risk factor forfemales in India Biotech “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsHuebner J Marienfeld S Abbenhardt C Ulrich C Muenstedt K Micke O Counseling patients on cancer diets a review of the literature andrecommendations for clinical practice Anticancer Res “Champ CE Palmer JD Volek JS WernerWasik M Andrews DW Evans JJ Targeting metabolism with a ketogenic diet during the treatment ofglioblastoma multiforme J NeuroOncol “Zuccoli G Marcello N Pisanello A Servadei F Vaccaro S Mukherjee P et alMetabolic management of glioblastoma multiforme using standard therapytogether with a restricted ketogenic diet case report Nutr Metab Servaes P Verhagen C Bleijenberg G Fatigue in cancer patients during andafter treatment prevalence correlates and interventions Eur J Cancer “Cohen C Fontaine K Arend R Soleymani T Gower B Favorable effects of aKetogenic diet on physical function perceived energy and food cravings inwomen with ovarian or endometrial Cancer a randomized Controlled TrialNutrients Breymeyer KL Lampe JW McGregor BA Neuhouser ML Subjectivemood and energy levels of healthy weight and overweightobesehealthy adults on highand lowglycemic load experimental dietsAppetite “TanShalaby JL Carrick J Edinger K Genovese D Liman AD Passero VA Modified Atkins diet in advanced malignanciesfinal results of a safetyand feasibility trial within the veterans affairs Pittsburgh healthcare systemNutr Metab Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Klement RJ Sweeney RA Impact of a ketogenic diet intervention duringradiotherapy on body composition I Initial clinical experience with sixprospectively studied patients BMC Res Notes Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Tóth C Clemens Z Halted progression of soft palate Cancer in a patienttreated with the Paleolithic Ketogenic diet alone a 20months followupAm J Med Case Rep “ Gibson AA Seimon RV Lee CM Ayre J Franklin J Markovic T Doketogenic diets really suppress appetite A systematic review and metaanalysis Obes Rev “Sremanakova J Sowerbutts A Burden S A systematic review of the use ofketogenic diets in adult patients with cancer J Hum Nutr Diet “Khodabakhshi A Akbari ME Mirzaei HR Kazemian E Kalantari K Kalamian M Effects of ketogenic diet for breast cancer treatment A protocol forrandomized controlled clinical trial J Biochem Technol “Khodabakhshi A Akbari ME Mirzaei HR MehradMajd H Kalamian MDavoodi SH Feasibility safety and beneficial effects of MCTbasedKetogenic diet for breast Cancer treatment a randomized controlled trialstudy Nutr Cancer Khodabakhshi A Akbari ME Mirzaei HR Seyfried TN Kalamian M DavoodiSH Effects of Ketogenic metabolic therapy on patients with breast Cancer arandomized controlled clinical trial Clin Nutr in press Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe European anization for Research and Treatment of Cancer quality oflife questionnaire EORTC QLQC30 translation and validation study of theIranian version Support Care Cancer “ Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe EORTC breast cancerspecific quality of life questionnaire EORTC QLQBR23 translation and validation study of the Iranian version Qual Life Res“ Champ CE Volek JS Siglin J Jin L Simone NL Weight gain metabolicsyndrome and breast cancer recurrence are dietary recommendationssupported by the data Int J Breast Ca

"Meningiomas are the most common primary central nervous system tumors Potential risk factorsinclude obesity height history of allergyatopy and autoimmune diseases but findings are conflicting This studysought to assess the role of the different risk factors in the development of meningioma in adolescentsyoungadultsMethods The cohort included Jewish men and women who had undergone compulsory physicalexamination between and at age to years prior to and independent of actual military enlistmentTo determine the incidence of meningioma the military database was matched with the Israel National CancerRegistry Cox proportional hazard models were used to estimate the hazard ratios for meningioma according to sexbody mass index BMI height and history of allergic or autoimmune diseaseResults A total of subjects females were diagnosed with meningioma during a followup of personyears Median age at diagnosis was ± years range “ On univariate analysis female sex p and height p were associated with risk of meningioma When the data were stratified by sex heightremained a significant factor only in men Spline analysis of the male subjects showed that a height of m wasassociated with a minimum disease risk and a height of meters with a significant riskConclusions This large population study showed that sex and adolescent height in males m wereassociated with an increased risk of meningioma in adulthoodKeywords Allergy Autoimmune disease Height Meningioma SexBackgroundMeningiomas are the most common primary centralnervous system tumors They originate from the meninges which are the membranous layers surrounding thebrain Most meningiomas “ are grade I benign“ are grade II atypical and “ are grade IIIanaplastic [] Benign meningiomas have a female predominance or which is not found in the more Correspondence matanbe4gmailcom1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva IsraelFull list of author information is available at the end of the aggressive types [] In the USA meningiomas werefound to be more common in blacks than in whites witha ratio of [] The risk of acquiring a meningiomaincreases with age The median age at diagnosis is years []The only established external nongenetic risk factorfor brain tumors is exposure to ionizing radiation []An Israeli study revealed abnormally high rates of meningioma in patients treated with lowdose radiation tothe scalp for tinea capitis during the 1950s [] Otherpotential risk factors include obesity height history ofallergyatopy and history of autoimmune diseases but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenZion Berliner BMC Cancer Page of the results are conflicting [“] Establishing risk factors for meningioma can help identify individuals whomight benefit from risk reduction strategies and possiblyearly screening methodsThe aim of the present study was to assess potentialrisk factors for the development of meningioma in adolescence and early adulthoodMethodsStudy populationIsraeli adolescents undergo a compulsory medicalexamination at age to assess their fitness for military service regardless of whether they are drafted ornot Arab and Orthodox Jewish females and malesand Druze females are exempted Together with thephysical examination sociodemographic and psychobehavioral data are collected and the medical historyis thoroughly reviewed using documents provided byeach subject™s primary care physician At the end ofthe process recruits are assigned a Functional Classification Code FCC that describes their medical status and occupational medical ranking The medicaldata and FCC are stored in the army™s main databasewhich was computerized in []The population for the present study was derived from subjects born in “ who underwentpreenlistment medical examination between whenthe database was computerized and at age “years Subjects with missing data on height and weightwere excluded n We also excluded subjects of North African and Asian origin born before many of whom had been exposed to radiation forthe treatment of tinea capitis after immigrating to Israelduring the 1950s [] These communities were laterfound to have a particularly high rate of meningioma []The final cohort consisted of subjects Fig Records were reviewed from the date of the initialmedical examination for military fitness to the date of afirst diagnosis of any cancer death or the predeterminedend of followup December Study variablesAtthe preenlistment medical examination demographic variables for each recruit were recorded in thearmy database as follows date of birth age at examination country of origin education socioeconomic status height weight and body mass index BMI Originwas defined by father™s country of birth or if the examinee™s father was born in Israel by paternal grandfather™scountry of birth and categorized as Europe includingNorth and South America Australia and SouthernAfrica Asia predominantly the Middle East Africaoverwhelmingly North Africa and Israel third or latergeneration Education was categorized as ‰ Fig Selection of the study populationand ‰¥ years of schooling Socioeconomic status wasdetermined by place of residence at the time of examination coded on a scale of “ and categorized intolow score “ middle score “ and high score “ [] Height and weight were measured by trainedmedics using a stadiometer and a beam balance with examinees barefoot and in underwear BMI was calculatedas weight in kilograms divided by height squared inmeters and categorized according to the WHO asunderweight kgm2 healthy weight “kgm2 overweight “ kgm2 and obese ‰¥kgm2 Height was categorized according to the Centersfor Disease Control and Prevention below 25th percentile 25th to 50th percentile 50th to 75th percentile and75th percentile and aboveCognitive function including language skills and intellectual performance [] was assessed by a general 0cBenZion Berliner BMC Cancer Page of intelligence test administered by trained personnel Thetest is scored on a 90point scale that is adjusted fromtime to time scores are categorized as low “medium “ and high “ []inflammatory bowel disease pemphigusMedical history was assessed according to the FCCslupus vascuAutoimmune diseases diabetes mellituslitisthyroiddisease celiac rheumatoid arthritis Addison disease andidiopathic thrombocyt ia purpura and allergic diseases asthma urticaria eczema allergic rhinitis atopicdermatitis allergic conjunctivitis and anaphylaxis weregrouped together for the present analysisAscertainment of meningioma incidenceTo determine the incidence of meningioma we matchedthe subjects who underwent the preenlistment medicalexamination during the study years to the Israel National Cancer Registry INCR a national populationbased registry established in In Israeli lawmandated the reportage of all diagnoses of malignant insitu and invasive borderline and certain benign brainand central nervous system tumors The estimated rateof reportage for solid tumors is which meets thestandards of the International Association of CancerRegistrieswwwhealthgovilPublicationsFilesICDC_365_EN_summarypd The INCR data includethe date of diagnosis site affected the InternationalClassification of Diseases code and the histologic description of the tumor according to the third edition ofthe International Classification of Diseases for OncologyICDO3 codes and At the time ofmatching the INCR had been updated until the end ofStatistical analysisCategorical variables are presented as number and percentage and continuous variables as mean and standarddeviation SD median 25th and 75th percentiles minimum and maximum were also calculated The association between risk factors and time to meningiomadiagnosis was assessed using Cox proportional hazardmodels hazard ratios HR confidence intervals CI and pvalues were calculated Log minus logfigures were inspected to confirm the proportionality ofthe hazard Crude rates were also determined Independent variables were initially entered individually into theCox model After sex and the interaction of sex andheight were found to be statistically significant separatemodels were established for men and women A Cox regression cubic spline function with three equally spacedknots positioned between the minimum and maximumvalues of height was fit to the data to estimate the heightvalue associated with minimum risk of meningioma inmen SASSTAT and SASGRAPH software version SAS Institute Inc Cary NC USA Other statistical analyses were performed with SPSS Statistics for Windowsversion IBM Armonk NY USA Twosided pvalues of ‰ were considered statistically significantResultsStudy populationThe baseline characteristics of the study population arepresented in Table The mean age at initial examination was ± years of the cohort was female The mean duration of followup was ± years median which represent in this study population a follow up of personyears The characteristics of the medical history of the subjects arepresented in the supplementary table Table 1SLinkage of the military database with the INCR yieldeda diagnosis of meningioma in of the subjects who underwent medical examination in to at age “ years grade I atypical anaplastic not specified and one patient with meningiomatosisTable The mean age at diagnosis ofmeningioma was ± years range “ andat the end of followup ± years median Univariate analysisOverall as expected meningiomas were more common infemales cases crude rate per personyears than in males cases crude rate per personyears p HR CI “ However there was no sex difference in the incidence for themore aggressive meningiomas atypical and anaplasticcrude rate per personyears for males andfemales On univariate analysis only sex and height weresignificantly associated with the risk of meningioma in thewhole study population p for both variables Afterstratification by sex height remained significant only inmales Table The risk of meningioma was minimalwhen height was up to m and statistically significantwhen height was greater than m Fig BMI was notassociated with an elevated risk of meningioma even whenanalyzed separately by sex Table Past medical history of asthma diabetes and otheratopic or autoimmune diseases was not associated withrisk of meningioma Even when autoimmune and allergic diseases were analyzed as a group there was no association with lower risk of meningioma Table andSupplemental Table When the subjects of African and Asian origin whowere excluded from the main analysis were included inthe cohort there was a significant interaction betweenperiod of birth “ vs “ and Asianand African origin representing the Middle East andNorth Africa as opposed to European and Israeli origin 0cBenZion Berliner BMC Cancer Page of Table Baseline characteristics of the study population total and by sexMaleCharacteristicsNumberBirth yearTotal““““LowMediumHigh years years years years“““ 25th percentile25th“50th percentile50th“75th percentile 75th percentileEuropeAsiaAfricaIsraelEuropeAsiaAfricaIsraelSocioeconomic statusEducationCognitive indexaBMI category Kgm2Height category CDC percentileCountry of birthOriginAge at time of medical examination yearsBMIHeight metersaRated on a 90point scaleFemaleNumberSDMeanSDTotalNumberMeanSDMeanThe conjoined effect of birth year and origin showedthat origin North Africa and Asia was significant onlyfor subjects born between and SupplementalTable DiscussionIn this nationwide populationbased study we analyzedthe association of the development of meningioma insubjects born between and with baseline variables obtained for the subjects at the average age of years As expected meningiomas were found to be associated with sex female and taller stature None of theother sociodemographic and medical variables assessedincluding BMI and a diagnosis of asthma or diabetes atage years was associated with an increased risk ofmeningioma 0cBenZion Berliner BMC Cancer Page of Table Meningioma type and rate total and by sexMeningioma typeMeningioma NOSMeningiomatosisGrade Atypical AnaplasticTotalPersonyearsNOS Not otherwise specifiedMalesNumberPer FemalesNumberPer TotalNumberPer It is well accepted that benign meningioma is morecommon in females than males but the sex predilectiondisappears with the more aggressive meningiomas []The female predominance might be explained by thefinding that meningiomas harbor receptors for estrogenand progesterone []We discovered an association between the risk ofmeningioma and height in men but not with BMI inmen or women The results of previous studies for thesetwo factors were conflicting A large Norwegian studyincluding million subjects found that height was associated with meningioma in both men and women butBMI was not [] whereas another study of postmenopausal females revealed an association of meningiomawith both BMI and height [] A metaanalysis of studies supported the correlation of BMI and meningioma It is worth noting that the Norwegian study exceeds the metaanalysis in size and power and that inthe Norwegian study a subgroup analyses for womenand men as well as different age groups was performedwithout finding convincing evidence of a strong association between overweight obesity and risk for meningioma [] In our study BMI was measured when thesubjects were years old much younger than theTable Univariate analysis association of potential risk factors with diagnosis of meningioma by sexVariablesMalesNCases Crude rate HR CILower UpperpCases Crude rate HR CILower UpperpFemalesNHeightHeight continuousBMIPercentile ““ Kgm2 Autoimmune diseasesa NoAllergic diseasesbAsthmaDiabetesYesNoYesNoYesNoYes aAutoimmune disease diabetes mellitus lupus vasculitis IBD pemphigus thyroid disease celiac rheumatoid arthritis Addison disease and idiopathic thrombocyt icpurpurabAllergic disease including asthma urticaria eczema allergic rhinitis atopic dermatitis allergic conjunctivitis and anaphylaxis 0cBenZion Berliner BMC Cancer Page of Fig Spline analysis in the men group showing the minimum risk for meningioma at a height of m and a statistically significant increase inthe risk for meningioma at heights taller than mstudies included in the metanalysis which might explainthe discordant results []Height has been associated with different types of cancer melanoma thyroid testis breast and lymphomaSuggested mechanism for the greater risk of meningioma in taller people is their higher levels of circulatinginsulinlike growth factors IGFs which may influencecell proliferation and tumor growth [] Moreoveroverexpression of IGFI and IGFII mRNA transcriptshas been demonstrated in meningioma [] Circulatinglevels of IGFs are highest during puberty They decreaserapidly in the third decade of life in the general population but seem to stay consistently higher in taller adults[] It is not clear why this association was evident onlyin males in our study maybe in women the influence ofthe hormonal status blurred the influence of the heightSeveral earlier studies reported an inverse associationbetween a history of allergic diseases including asthmaand meningioma [ ] However this finding wasnot supported by others [ ] We failed to demonstrate an association between meningiomas and allergicdiseases including asthma urticaria eczema allergicrhinitis atopic dermatitis conjunctivitis and anaphylaxisand allergy to beesSimilarly a recent study reported an inverse association between hyperglycemia and the risk of meningioma [] whereas another found a positive associationwith a history of diabetes mellitus [] In the presentstudy diabetes was not associated with the risk of meningioma This was true for other autoimmune diseasesas welllimitationThis analysis also has certain limitations The followup period in this study was limited to years such thatthe study population was still young when the studyended Subsequently the median age of those who developed meningioma in our study was younger than themedian age of patients with meningioma in the generalpopulation [] With a more extensive followup wemight find more latent tumor growths that could potentially increase or shift the incidence of intracranial neoplasms Anotherisunderreporting of meningiomas that are diagnosed onlyaccording to radiographic findings without histologicalfindings As it is well known that in some cases meningiomas diagnosed radiographically mayjust befollowed by repeat scanningcohortits prospectivepopulationbased design large sample size high degreeof completeness of the cancer registry data throughoutthe study period and the ability to carefully control forpotential confounders such as exposure to radiation Itshould be noted that in a study that was published recently and examined the same cohort the median heightremained almost stable during the study period theStrengths of ourofthestudyinclude 0cBenZion Berliner BMC Cancer Page of median height of males increased by cm and that offemales remained stable despite environmental socialand nutritional changes []ConclusionThis large populationbased study showed that sex female and tall stature in adolescent males was associatedwith an increased risk of meningioma in adulthoodSupplementary informationSupplementary information accompanies this paper at doi101186s12885020072924Additional file Supplementary Table Medical historycharacteristics of the study populationAdditional file Supplementary Table Univariate analysisassociation of potential risk factors with diagnosis of meningioma by sexAdditional file Supplementary Table Interaction between birthperiod and origin whole population adjusted for sexAbbreviationsCNS Central nervous system BMI Body mass index FCC FunctionalClassification INCR Israel National Cancer Registry ICDO InternationalClassification of Diseases for Oncology SD Standard deviation HR Hazardratio CI Confidence interval IGF Insulinlike growth factorAcknowledgmentsNot applicableAuthors™ contributionsMBZB and SYK analyzed the preliminary database extracted the relevantinformation to allow hypothesis testing and prepared the manuscript andtables Statistical analysis and figure preparation were performed by LHK andED HL LKB YL AH JM and GT participated in the preliminary preparationand conceptual design and revised the final manuscript ABA OG AK and TSreviewed the neurological proof of concept and revised the final manuscriptand supplementary materials All authors read and approved the finalmanuscriptFundingThe study was funded by the Israel Cancer Association by the Lillia andJacob Alther donation financial support without any role in the manuscriptpreparationAvailability of data and materialsThe datasets used during this study are available from the correspondingauthor on reasonable requestEthics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional andor nationalresearch committee and with the Helsinki declaration and its lateramendments or comparable ethical standards The study was approved bythe IDF Israel Defense Forces Medical Corps Institutional Review Boardwhich waived the requirement for informed consent because the data usedwere obtained from medical records without patient participation referencenumber “Consent for publicationNot applicableAuthor details1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva Israel 2Department of GastroenterologyHadassah University Hospital “ Ein Kerem Jerusalem Israel 3Sackler Facultyof Medicine Tel Aviv University Tel Aviv Israel 4Braun School of PublicHealth and Community Medicine Hadassah University Hospital “ Ein KeremJerusalem Israel 5Israel Center for Disease Control Israel Ministry of HealthRamat Gan Israel 6School of Public Health University of Haifa Haifa Israel7Department of Neurosurgery Rabin Medical Center “ Beilinson HospitalPetach Tikva Israel 8Medical Corps Israel Defense Forcesand Department ofMilitary Medicine Hebrew University of Jerusalem Faculty of MedicineJerusalem Israel 9Institute of Endocrinology and Talpiot Medical LeadershipProgramSheba Medical Center Tel Hashomer IsraelReceived April Accepted August ReferencesOstrom QT Gittleman H Fulop J Liu M Blanda R Kromer C Wolinsky YKruchko C BarnholtzSloan JS CBTRUS statistical report primary brain andcentral nervous system tumors diagnosed in the United States in NeuroOncology 201517Suppl 4iv1“iv62 doi101093neuoncnov189Claus EB Bondy ML Schildkraut JM Wiemels JL Wrensch M Black PMEpidemiology of intracranial meningioma Neurosurgery “doi10122701neu000018828191351b9Braganza MZ Kitahara CM Berrington de González A Inskip PD Johnson KJRajaraman P Ionizing radiation and the risk of brain and central nervoussystem tumors a systematic review NeuroOncology “doi101093neuoncnos208 Modan B Baidatz D Mart H Steinitz R Levin SG Radiationinduced headand neck tumours Lancet “ doi101016s0140 Wiedmann MKH Brunb C Di Ieva A Lindemann K Johannesen TBVatten L Helseth E Zwart JA Overweight obesity and height as risk factorsfor meningioma glioma pituitary adenoma and nerve sheath tumor alarge populationbased prospective cohort study Acta Oncol “ doi1010800284186X20171330554Johnson DR Olson JE Vierkant RA Hammack JE Wang AH Folsom ARVirnig BA Cerhan JR Risk factors for meningioma in postmenopausalwomen results from the Iowa Women's health study NeuroOncology“ doi101093neuoncnor081 Michaud DS Bové G Gallo V Schlehofer B Tjønneland A Olsen A OvervadK Dahm CC Teucher B Boeing H Steffen A Trichopoulou A Bamia CKyrozis A Sacerdote C Agnoli C Palli D Tumino R Mattiello A BuenodeMesquita HB Peeters PH May AM Barricarte A Chirlaque MD DorronsoroM José Sánchez M Rodríguez L Duell EJ Hallmans G Melin BS Manjer JBquist S Khaw KT Wareham N Allen NE Travis RC Romieu I Vineis PRiboli E Anthropometric measures physical activity and risk of glioma andmeningioma in a large prospective cohort study E Cancer Prev Res Phila“ doi10115819406207CAPR110014Niedermaier T Behrens G Schmid D Schlecht I Fischer B Leitzmann MFBody mass index physical activity and risk of adult meningioma andglioma a metaanalysis Neurology “ doi101212WNL0000000000002020Brenner AV Linet MS Fine HA Shapiro WR Selker RG Black PM Inskip PDHistory of allergies and autoimmune diseases and risk of brain tumors inadults Int J Cancer “ doi101002ijc10320 Wang M Chen C Qu J Xu T Lu Y Chen J Wu S Inverse associationbetween eczema and meningioma a metaanalysis Cancer Causes Control“ doi101007s1055201198086 Gal R The selection classification and placement process in a portrait ofthe Israeli soldier Westport CT Greenwood Press p “ YustKatz S Bar Oz A Derazne E Katz LH Levine H KeinanBoker L Amiel ACompeting interestsThe authors declare that they have no financial or nonfinancial competinginterestsKanner A Laviv Y Honig A Shelef I Siegal T Twig G Kark J Echoes fromthe past changing associations between brain tumors and ethnicity JNeurol Sci doi101016jjns2019116552 [Epubahead of print]Israel Central Bureau of Statistics Characterization and classification of localauthorities by the socioeconomic level of the population Jerusalem Israelcentral Bureau of Statistics 0cBenZion Berliner BMC Cancer Page of Twig G Gluzman I Tirosh A Gerstein HC Yaniv G Afek A Derazne E Tzur DKarasik A Gordon B Fruchter E Lubin G Rudich A CukiermanYaffe TCognitive function and the risk for diabetes among young men DiabetesCare Nov37112982“ doi102337dc140715 Guevara P EscobarArriaga E SaavedraPerez D MartinezRumayor A FloresEstrada D Rembao D Calderon A Sotelo J Arrieta O Angiogenesis andexpression of estrogen and progesterone receptors as predictive factors forrecurrence of meningioma J NeuroOncol “ doi101007s110600172662y Gunnell D Oliver SE Donovan JL Peters TJ Gillatt D Persad R Hamdy FCMeal DE Holly JMP Do heightrelated variations in insulinlike growthfactors underlie the associations of stature with chronic diseases J ClinEndocrinol Metab “ doi101210jc2003030507 Zumkeller W Westphal M The IGFIGFBP system in CNS malignancy MolPathol “ Crowe FL Key TJ Allen NE A crosssectional analysis of theassociations between adult height BMI and serum concentrations of IGFIand IGFBP1 and ˆ’ in the European prospective investigation intocancer and nutrition EPIC Ann Hum Biol “ doi BergBeckhoff G Schüz J Blettner M Münster E Schlaefer K Wahrendorf JSchlehofer B History of allergic disease and epilepsy and risk of glioma andmeningioma INTERPHONE study group Germany Eur J Epidemiol “ doi101007s1065400993556Schneider B Pülhorn H Röhrig B Rainov NG Predisposing conditions andrisk factors for development of symptomatic meningioma in adults CancerDetect Prev “ doi101016jcdp200507002Linos E Raine T Alonso A Michaud D Atopy and risk of brain tumors ametaanalysis J Natl Cancer Inst “ doi101093jncidjm170 Bernardo BM Orellana RC Weisband YL Hammar N Walldius G MalmstromH Ahlbom A Feychting M Schwartzbaum J Association betweenprediagnostic glucose triglycerides cholesterol and meningioma andreverse causality Br J Cancer 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"The DICER algorithm (24) seeks one pair of linked modules at a time. A pair of modules is defined as linked if the sum of weights WG between them is high enough. We call the approach of DICER ˜local™ as it finds one module pair at a time. The algorithm of Ulitsky et al. (17) aims to maximize the ˜global score™ namely the total sum of scores within modules in H plus the sum of scores of links in G. In addition to increasing the global score links between modules are accepted only if they pass a statistical significance test. We call the second approach ˜global™. Both methods identify the links and the modules simultaneously.D demonstrates the differences between the local and global approaches. Assume that in both graphs edge weights are 1 non-edge weights are ?1 and that the local approach uses a threshold of 0 on the sum of WG weights between two modules for reporting a link. In both approaches modules are clusters of nodes with high density in H. According to both approaches module 1 is linked to module 2: the local score is 4 (8 edges and 4 non-edges) the global analysis P-value for linkage is <0.05 and the total score for the module pair is 13 (module score 6 + 3 + link score 4). The sum of WG weight between modules 2 and 3 is ?4 (10 edges and 14 non-edges) and the local method rejects that link. However the global approach will also link module 2 and 3: the linkage P-value is significant (P = 0.039) and adding this link will improve the global map score to 24 [13 for the (12) pair +15 for module 3“4 for the (23) link]. This example illustrates the advantage of the global approach on sparse graphs in which large modules are not expected to be densely interconnected.AlgorithmsWe conducted a systematic study and developed further a family of two-phase algorithms for module map detection that find an initial solution (possibly consisting of many small modules) and then improve it. We call algorithms for the first phase initiators and algorithms for the second phase improvers. For simplicity we describe the algorithms assuming that edges with positive weight are considered heavy. For unweighted graphs we assume edge weights to be 1 and non-edge weights to be ?1. For weighted graphs all node pairs (edges) have weights so there are no non-edges.InitiatorsWe tested five different initiators: (i) DICER (24) which finds one pair of linked modules at a time (ii) hierarchical clustering of the graph H (25) which finds a set of modules (iii) a greedy node addition algorithm for finding modules in H (iv) DICERk a variant of DICER wherein the minimum module size is set to k and (v) an algorithm based on enumeration of maximal bicliques in G using an exhaustive solver (2627) followed by the cleaning process of DICER. We call the latter algorithm MBC-DICER see Supplementary Text and Supplementary Figure S1 for a full description of all initiators. Each initiator creates an initial module set but modules in the map constructed by clustering algorithms are not necessarily linked.ImproversThe ˜local improver™ (24) extends module map links by either adding a single node to a module or by merging two module map links. One drawback of this approach is that it cannot create new modules that are not represented in the initial solution. Another disadvantage is that it cannot merge a module whose two parts are linked to different modules that are unlinked. See Supplementary Figure S2 for examples."

" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimer™s disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats “ catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnÎ interferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinson™s disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnÎ further elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated œoxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide human™s nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino virus™s infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap ˜n™ collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies “ neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra

"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adults™population of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [“] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [“] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients™ intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at ˆ’ °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients™ intakeUsual Macronutrients™ intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants™ physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrients™intake and physical activity between different FTO genotypes The post hoc Tukey™s test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrients™protein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [“] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ‰¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableˆ’Calorieˆ’ˆ’ˆ’ˆ’ATˆ’ˆ’ˆ’ˆ’P valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele ß ˆ’ kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrients™intakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [“] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients™ intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3“ and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors™ contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet “Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States “ JAMA “Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol “Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab “Fredriksson R Hagglund M Olszewski PK Stephansson O Jacobsson JAOlszewska AM Levine AS Lindblom J Schiöth HB The obesity gene FTO isof ancient origin upregulated during food deprivation and expressed inneurons of feedingrelated nuclei of the brain Endocrinology May “Doaei S Hajiesmaeil M Aminifard A MosaviJarrahi SA Akbari MEGholamalizadeh M Effects of gene polymorphisms of metabolic enzymeson the association between red and processed meat consumption and thedevelopment of colon cancer a literature review J Nutritional Sci Doaei S Kalantari N Mohammadi NK Izadi P Gholamalizadeh M EiniZinabH Salonurmi T Jarrahi AM Rafieifar S Janipoor R Sadeghypor M Upregulation of FTO gene expression was associated with increase in skeletalmuscle mass in overweight male adolescents Arch Med Sci Sep155Hakanen M Raitakari OT Lehtimäki T Peltonen N Pahkala K Sillanmäki LLagstrom H Viikari J Simell O Ronnemaa T FTO genotype is associatedwith body mass index after the age of seven years but not with 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Jarrahi AM Rafieifar S Azizi Tabesh GRahimzadeh G Gholamalizadeh M Goodarzi MO Interactions between macronutrients™ intake FTO and IRX3 gene expression and FTO genotype in obeseand overweight male adolescents Adipocyte Jan “ Olszewski PK Fredriksson R Olszewska AM Stephansson O Alsiö JRadomska KJ Levine AS Schiöth HB Hypothalamic FTO is associated withthe regulation of energy intake not feeding reward BMC Neurosci Dec1011“ Razquin C Martinez JA MartinezGonzalez MA A 3year intervention with aMediterranean diet modified the association between the rs9939609 genevariant in FTO and body weight changes Int J of Obesity “ McTaggart JS Lee S Iberl M Church C Cox RD Ashcroft FM FTO isexpressed in neurones throughout the brain and its expression is unalteredby fasting PLoS One 2011611e27968 httpsdoi101371journalpone0027968Stratigopoulos G Padilla SL LeDuc C A Watson E Hattersley AT McCarthyMI Zeltser LM Chung WK Leibel RL Regulation of FtoFtm gene expressionin mice and humans Am J Physiol Integr Comp Physiol 2008294R1185“ Wang P Yang FJ Du H Guan YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May ““ Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3“ N Engl J Med Sep “ Wardle J Carnell S Haworth CM Farooqi IS O™Rahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab “ httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E O™Daly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspolymorphisms rs1421085 rs17817449 and rs9939609 in exercisetrainedmen and women the effects of a 4week hypocaloric diet J Int Soc SportsNutr Dec Blundell JE Lawton CL Cotton JR Macdiarmid JI Control of humanappetite implications for the intake of dietary fat Annu Rev Nutr “Ludwig DS The glycemic index physiological mechanisms relating toobesity diabetes and cardiovascular disease Jama “Sonestedt E Roos C Gullberg B Ericson U Wirfält E OrhoMelander M Fatand carbohydrate intake modify the association between genetic variationin the FTO genotype and obesity Am J Clin Nutr Sep “Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relativevalidity of food group intake in a food frequency questionnaire developedfor the Tehran lipid and glucose study J Epidemiol “ Azar M Sarkisian E Food composition table of Iran National Nutrition andfood research institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes “ Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455“ Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May “ Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov “ Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul “ Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov “Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript 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t he gut is the largest immune an of the humanbody and also an important target an for variouskinds of stress caused by severe insults such as infectiontrauma and shock12 these stresses are considered to haveinstitution at which the work was performed department oftraumatology and acute critical medicine osaka universitygraduate school of medicine yamadaoka suita osaka japancorresponding yasutaka nakahori md division of trauma andsurgical critical care osaka general medical center bandaihigashi sumiyoshi ward osaka japan emailp_olysyahoocojpreceived apr accepted jul funding informationno funding information providedan important role in promoting infectious complications andmultiple an dysfunction syndrome from the viewpointsof deteriorated intestinal epithelium the immune systemand commensal bacteria gut dysfunction is now recognizedas a cause for the promotion of diseases34lipids have been a focus not only as energy sources butalso as immunemodulating substrates5 unlike long andmediumchain fatty acids shortchain fatty acids scfaswhich mainly consist of acetate propionate and butyratewith two to four carbon atoms are principally derived fromthe fermentation of carbohydrates and amino acids by anaerobic microanisms shortchain fatty acids are usedmainly by intestinal epithelial cells as energy substrates andthey ‚uence the motility of the intestinal tract and increaseintestinal blood flow we previously reported on altered gutflora and fecal anic acids in critically ill patients andshowed that these patients had significantly lower levels of the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicinethis is an open access under the terms of the creative commons attributionnoncommercial licensewhich permits use distribution and reproduction in any medium provided the original work is properly cited andis not used for commercial purposes of 0c of y nakahori acute medicine surgery 20207e558fecal anic acids especially scfas than healthy volunteers67despite the evidence implicating the importance ofscfas in the gut the effect of scfas on prognosis in critically ill patients has not yet been clarified in this study wemeasured eight kinds of fecal anic acidsincludingscfas in critically ill patients and investigated the effect ofscfas on their prognosismethodspatientst his retrospective study enrolled patientswho i fulfilled the criteria of systemic ‚ammatoryresponse syndrome sirs according to the american college of chest physicians and the society of critical caremedicine8 ii had a serum creactive protein crp levelgreater than mgdl iii were admitted to the departmentof traumatology and acute critical medicine osakauniversity graduate school of medicine osaka japan during the period november “january iv were treated in the intensive care unit for more than days we usedcrp ‰¥ mgdl to focus on cases with severe ‚ammationbecause crp is mainly used as a marker of ‚ammationwe excluded patients from whom we could not collect anyfecal samples during hospitalization continuous enteralfeeding was initiated with kcalday and graduallyincreased depending on the patient™s condition we usedimpact novartis nutrition minneapolis mn usa as anenteral nutritional product for all patients in our intensivecare unit and used “ kcalkg ideal body weight as thegoal for the administered dose fourteen healthy volunteersprovided fecal samples as controls for examinations of fecalanic acids concentrations this study was approved bythe institutional review board of osaka university approval no and informed consent was obtained fromeach patient™s familyfecal samples and determination of fecalanic acid concentrationswe collected fecal samples serially from all patients thefecal samples were transported at 00°c to the yakult central institute tokyo japan we measured eight kinds ofanic acid in the feces by highperformance liquid chromatography feces were homogenized in ml distilledwater the homogenate was then placed in an eppendorftube and centrifuged at g for min at °c a mixture of ml of the resulting supernatant and ml of moll perchloric acid was mixed well in a glass tubeand allowed to stand at °c for h the suspension wasthen passed through a filter with a pore size of lmnihon millipore tokyo japan the sample was analyzedfor anic acids by highperformance liquid chromatography as previously described9 using a waters conductivity detector waters milford ma usa equipped withtwo columns shodex kc811 showa denko tokyojapan the concentrations of fecal anic acids were calculated with the use of external standards the reproducibilityand stability of these measurements were shown previously10 we retrospectively evaluated the minimum andmaximum value of each fecal anic acid measured duringhospitalization and determined prognostic factors by classification and regression tree cart analysissurveillance and definition of complicationsbacterial infection was diagnosed in accordance with thedefinitions of the centers for disease control and prevention11 body temperature was measured continuouslysurveillance cultures from blood and sputum were undertaken routinely for each patient in cases of suspected infectionand computedtomography scanning were carried out as necessary bacteremia was defined as a positive blood culturelaboratory testingchest xraystatistical analysiscontinuous variables expressed as the median 25th and 75thpercentiles were compared by the mann“whitney utestcategorical variables expressed as number were comparedby the v2test unless the expected counts in any of the cellswere below in which case the fisher exact test was usedwe used cart analysis which is a binary recursive partitioning using nonparametric approaches to identify keyfecal anic acids and their cutoff values for mortality12we also used multivariate logistic regression analysis toquantitatively evaluate the effect of the covariates that weresuggested by cart analysisthe most important prognostic factors were selected anda predictive model was developed as follows first the minimum and maximum values of each fecal anic acid wereselected from all patients the maximum value was the highest value and the minimum value was the lowest value measured during hospitalization second the mann“whitney utest was applied to discover variables with potential prognostic value and covariates with p were excludedfinally cart was used to create a tree using the minimumand maximum data values of all fecal anic acids theeffects of the minimum and the maximum values of the fecalanic acids that were selected as key prognostic factors by the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of table characteristics of critically ill patients included in this studytotal n survivors n nonsurvivors n pvalueage yearssex maleicu stay daysapache ii score on admissionorigins of sirssepsistraumaburnunknownbacteremianumber of antibiotic typesduration of antibiotic use days “ “ “ “ “ “ “ “ “ “ “ “ “ “ “categorical values are expressed as number continuous values are expressed as median 25th“75th percentiles bold values indicatestatistical significanceapache acute physiology and chronic health evaluation icu intensive care unit sirs systemic ‚ammatory response syndromecart analysis and age sex and acute physiology andchronic health evaluation apache …± score on admission were analyzed with the multivariable logistic regressionmodelsall reported pvalues were twosided and a pvalue of was considered to indicate statistical significance allstatistical analyses were undertaken using ibm spss statisticsversion armonk ny usaresultspatient characteristicsc haracteristics of the patients are shown intable there were survivors and nonsurvivors the two groups did not differ significantly in termsof sex or apache ii score age in the nonsurvivors wassignificantly older than that in the survivors p the principal origin of sirs was sepsis in of the patients in the two groups significantly more types ofantibiotics were used in the nonsurvivors than in the survivors during hospitalization p and the durationof antibiotic use in the nonsurvivors was significantlylonger than that in the survivors p the incidenceof bacteremia was significantly higher in the nonsurvivorsthan that in the survivors vs p anic acid profilestiming of fecal sample collection is shown in table wecollected fecal samples for scfa analysis fecalsamples were collected evenly throughout hospitalization inboth the survivors and the nonsurvivors the minimum andmaximum values of each fecal anic acid and the resultsof univariate analysis are shown in tables and in termsof the minimum values total anic acids acetate propionate and isovaleric acid were significantly decreased in thenonsurvivors in terms of the maximum values lactate formic acid and succinic acid were significantly increased inthe nonsurvivors otherwise isovaleric acid and acetatewere significantly decreased in the nonsurvivorsadditionally cart and multivariate logistic regressionanalyses of the predominant covariates were carried out fortable timing of fecal sample collection in critically illpatientssurvivorsnonsurvivorstotalweek week week week week week week week week week weekstotal the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table minimum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvaluetotal anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 ues indicate statistical significancetable maximum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvalue 06 06 06 06 06 06 06 06 06 total anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 ues indicate statistical significancethe minimum and maximum values of the fecal anicacids for the minimum values the primary split variablewas determined to be the minimum value of propionate andthe cutoff value was lmolg of feces the secondarysplit variable was determined to be the minimum value ofacetate and the cutoff value was lmolg of feces themortality in each partition and the diagnostic characteristicsof this tree are shown in figure for the maximum values of fecal anic acids the primary split variable was determined to be the maximumvalue of lactate and the cutoff value was lmolg offeces the secondary split variable was determined to bethe maximum value of formic acid and the cutoff valuewas lmolg of feces the mortality in each partitionand the diagnostic characteristics of this tree are shown infigure the diagnostic characteristics of the tree for minimumvalue showed a sensitivity of and specificity of whereas those ofthe tree for maximum valueshowed a sensitivity of and specificity of for the minimum values of fecal anic acids multivariate logistic regression analysis revealed that age and theminimum values of propionate and acetate were significantprognostic factors for mortality table for the maximumvalues age and maximum lactate were identified as significant prognostic factors for mortality table discussiont his study provides the first in vivo evidence toour knowledge that the altered balance of fecal anicacids is associated with mortality in critically ill patients arecent study has shown that scfas particularly acetate andpropionate bind the gproteincoupled receptor gpr43in adipose tissue stimulation of gpr43 by scfas was necessary for the anti‚ammatory responses because gpr43deficient mice showed exacerbated ‚ammation in modelsof colitis arthritis and asthma the scfa“gpr43 interactions profoundly affected intestinal and systemic ‚ammatory responses13 the low concentrations of acetate andpropionate observed in nonsurvivors in the present studycould reduce the anti‚ammatory effect fukuda 14showed that acetate produced by protective bifidobacteriaimproves intestinal defense mediated by epithelial cells andthereby protects the host against lethal infection furthermore asahara 15 showed a positive correlation betweenfecal acetic acid level and tightjunctionrelated gene expression in the intestinal epithelium in their experiment using aninfected mouse modelthe maximum values of lactate and formic acid were alsoselected as prognostic factors fig our group previouslyshowed that when patients had severely acidic feces fecallactate succinic acid and formic acid were significantlyincreased over those of patients with normal feces we alsoshowed that abnormal fecal ph was associated with the incidence of bacteremia and mortality16 the increase of lactateand formic acid can make feces severely acidic and severelyacidic feces could injure intestinal epithelial cells and worsen the patient™s condition the present results indicate thatnot only a lack of scfas but also an accumulation of lactateand formic acid might be associated with disruption of thegastrointestinal environmentin critically ill conditionswhich leads to bacterial translocationthere was a significant difference between the two groupsin age number of antibiotic types and duration of antibioticuse in this study previous studies have suggested thatchanges in the composition of bacterial species and genes in the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of fig charts showing mortality in critically ill patients partitioned by the minimum value of fecal anic acids using classificationand regression tree analysis min minimumfig charts showing mortality in critically ill patients partitioned by the maximum value of fecal anic acids using classificationand regression tree analysis max maximum the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table results of multivariate logistic regression analysis in critically ill patients using the minimum value of fecal anic acidscoeff bsepvalueodds ratio confidence intervallower limitupper limitmortalityagesexapache iipropionate minacetate min 00 00 00bold values indicate statistical significanceapache acute physiology and chronic health evaluation coeff b coefficient min minimum se standard errortable results of multivariate logistic regression analysis in critically ill patients using the maximum value of fecal anic acidscoeff bsepvalueodds ratio confidence intervallower limitupper limitmortalityagesexapache iilactate maxformic acid max 00bold values indicate statistical significanceapache acute physiology and chronic health evaluation coeff b coefficient max maximum value se standard errorthe intestinal flora occur with aging17 it is also reported thatthe genes related to scfa production were lost from theintestinal flora and that glycolytic ability was decreased withaging18 these changes could be one of the reasons whyaging was detected as an important factor related to mortality in the present study there is no doubt that antibiotics areimportant butthe intestinal florasome systemically administered antibiotics are excreted inthe bile and secreted in the upper gastrointestinal tract andupon reaching the colon they cause serious damage to theintestinal flora19 shortchain fatty acids could also bereduced with the destruction of the intestinal flora whichmight have worsened the prognosis20they adversely affectshortchain fatty acids are mainly used by intestinalepithelial cells as energy substrates and some are absorbedinto the portal flow to the liver and used as systemic energysources they can also ‚uence the motility of the intestinaltract absorption of electrolytes and pancreatic secretion andcan increase intestinal blood flow in the present study totalanic acids including scfas were significantly decreasedin the critically ill patients compared with those in thehealthy controls data not shown one reason would be thealtered gut flora that produce scfas the commensal gutflora in humans plays an essential role in homeostasis andprotection from injury in the gut2122 under critically illconditions it is difficult to maintain normal gut flora6 notonly does the stress derived from diseases such as traumaand burns affect the balance of the gut microbiota but alsothe stress caused by the treatment with various therapeuticagents such as histamine h2 receptor blockers catecholamines and broadspectrum antibiotics can ‚uence the gutflora we reported that critically ill patients had to times fewer total anaerobes bifidobacterium andlactobacillus and times greater staphylococcus whencompared with counts in healthy volunteers6 these dataindicate that the balance of gut flora is significantly disturbed in critically ill patients the concentrations of fecalanic acids decreased significantly in the nonsurvivors asshown in table and we believe thatthis was likelybecause the number of good bacteria bifidobacterium andlactobacillus had decreased similarly the cause of thereduction of shortchain fatty acids in the present study may the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of have been the reduction of the scfaproducing bacteriasuch as clostridium eubacterium peptococcusandfusobacterium2324synbiotics are the combination of probiotics and prebiotics the clinical effects of synbiotics have been reported inthe fields of pediatric surgery abdominal surgery and intensive care25 in our preliminary report critically ill patientstreated with synbiotics maintained the necessary amounts offecal anic acids including scfas and had fewer incidences of enteritis pneumonia and bacteremia than thosewithout synbiotics26 these reports suggest that the maintenance of gut flora and scfas would be a promising intestinal therapy synbiotics were given to patients in thisstudy in the synbioticstreated group maximum values offecal anic acids acetic acid propionic acid succinicacid lactic acid and formic acid increased significantly butminimum values did not increase only the minimum valueof lactic acid decreased significantly however there wasno fixed protocol for the treatment with synbiotics in thisstudy the treatment start date was variable and the averagehospital day of starting treatment was day “ so itwas difficult to evaluate the effects of synbiotics in thisstudy evaluating the impact of synbiotics on fecal anicacids and prognosis in critically ill patients will be a futuretaskour study has some limitations this was a retrospectivestudy and thus the timing of feces sampling was differentbetween patients the number of samples was too small tomake conclusions about the temporal patternsin conclusion the minimum values of fecal acetic acidand propionate in the nonsurvivors were significantly lowerthan those in the survivors the altered balance of fecalanic acids was associated with mortality in critically illpatients thus the maintenance of scfas could be a targetfor future intestinal therapyacknowledgementsw e thank all the staff of the department of traumatology and acute critical medicine osakauniversity school of medicinedisclosureapproval of the research protocol this study was approvedby the institutional review board of osaka universityinformed consent informed consent was obtained from eachpatient™s familyregistry and the registration no of the studytrial naanimal studies naconflict of interest nonereferences meddings j the significance of the gut barrier in disease gut “ macfie j o™boyle c mitchell cj buckley pm johnstoned sudworth p gut origin of sepsis a prospective studyinvestigating associations between bacterialtranslocationgastric microflora and septic morbidity gut “ clark ja coopersmith cm intestinal crosstalk a new paradigm for understanding the gut as the motor of critical illness shock “ deitch ea bacterial translocation or lymphatic drainage oftoxic products from the gut what is important in humanbeings surgery “ kreymann kg berger mm deutz ne espen guidelines on enteral nutrition intensive care clin nutr “ shimizu k ogura h goto m altered gut flora andenvironment in patients with severe sirs j trauma “ yamada t shimizu k ogura h rapid and sustainedlongterm decrease of fecal shortchain fatty acids in criticallyill patients with systemic 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a regulation of ‚ammatory responses by gut microbiota and chemoattractantreceptor gpr43 nature “ fukuda s toh h hase k bifidobacteria can protectfrom enteropathogenic infection through production of acetate nature “ asahara t takahashi a yuki n kaji r takahashi tnomoto k protective effect of a synbiotic against multidrug the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558resistant acinetobacter baumanniimodel antimicrob agents chemother “in a murine infection guarner f malagelada jr gut flora in health and diseaselancet “ osuka a shimizu k ogura h prognostic impact of gill sr pop m deboy rt metagenomic analysis of thefecal ph in critically ill patients crit care r119human distal gut microbiome science “ o™toole pw jeffery ib gut microbiota and aging science macfarlane s macfarlane gt regulation of shortchain fatty “ rampelli s candela m turroni s functional 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pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [“] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [“] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative real‘time polymerase chain reaction qRT‘PCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne„¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ˆ’ΔΔCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of × cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit‘ CCK8 assayMM cells were plated in 96well plates at the density of × cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur Becton“Dickinson San Jose CA USA and analyzed using Cell Quest software Becton“DickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur Becton“Dickinson and analyzed using Cell Quest software Becton“DickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dual‘luciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturer™s instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNA‘pull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturer™s instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Student™s t test or oneway analysis of variance ANOVA followed by Tukey™s test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearman™s correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells What™s more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR‘‘3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if œUGC UGG  in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its œUGC UGG  sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR e“k MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR‘‘3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearman™s coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR‘‘3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearman™s correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR‘‘3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells a“i MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5b“h The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR‘‘3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearman™s coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells a“i MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCs‘generated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07e“k sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis a“c Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins d“f The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 What™s more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings

predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

colorectal cancer crc is a malignant tumor in the gastrointestinal tract and it arises from theinner wall of the large intestine the colon crc is the third most common cancer worldwideaccounting for roughly million new cases per year and ˆ¼ deaths per year whichmakes it the fourth most common cause of cancerrelated death globally and remains a hugechallenge “ in order to identify eï¬ective molecular targets for crc diagnosis and potentialedited byzhonghua taofudan university shanghai cancercenter chinareviewed byshengli liuniversity of texas health sciencecenter at houston united statesrossano lattanziouniversity of studies g d™annunziochieti and pescara italycorrespondencejianhua wangwangjianhuaman163comyansong pudoctor_puyahoocom these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived september accepted june published august citationliu y cao j zhu yn ma ymurtaza g li y wang jh and pu ys c1222c deletion in exon ofabl1 is involved in carcinogenesisand cell cycle control of colorectalcancer through irs1pi3kaktpathway front oncol 103389fonc202001385frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerinterventions for crc therapy indepth studies on the regulatorymechanism of crc progression should be conductedthe pathogenesis of crc accompanies with genetic orepigenetic changes numerous genes and pathways such aswnt tgf egfr“ras erk“mapk pi3k and p53 havebeen demonstrated to be associated with crc “ abl1a protooncogene of cabl encodes a nonreceptor tyrosinekinase plays an important role in carcinogenesis regulatingcell adhesion proliferation diï¬erentiation and apoptosis studies have characterized abl1 as an oncogene thatpromotes breast cancer cell proliferation and induces anchorageindependent growth under p53 deficiency in breast cancercells “ craig reported that inhibition of abl1by imatinib reduced the proliferation of lymphoma cells andprevented tumor formation in mice however the roleand mechanism of abl1 in crc development and progressionremain largely unclearthe aim of this study was to elucidate the role of abl1using highthroughput dna sequencing technology to obtaininformation on colon cancer gene mutation we analyzedthe variation in the expression of abl1 among patients withcrc and in crc celllines we additionally determinedthe eï¬ect of downregulating abl1 on the proliferation cellcycle progression and apoptosis of crc cells further theeï¬ects of knockout of abl1 in tumor and the molecularmechanisms of activated and suppressed downstream signalingpathways were assayed to elicit the mechanisms involved incrc carcinogenesismaterials and methodsadmitted atclinicalpathological data ofpatients and samplesthefortyeight patients with crc wereshaanxi people™s hospitalshaanxi china colorectalcancer was confirmed by histopathology or biopsy basedon which thesubjectswere evaluated formalxed paraffinembedded ffpetissues were used as the study material tumor contentsin the ffpe tissues werethedepartment of histopathology shaanxi people™s hospitaland only ffpe tissue blocks with tumor contentwere qualified the study was approved by the ethicscommittee of shaanxi people™s hospital written informedconsent was obtained from allsubjects participating inthis studythethoroughlychecked atcell culture and rna interferencecrc cell lines ncm460 lovo sw620 sw480 and hct116were purchased from the cell bank of the chinese academyof sciences shanghai china the cells were cultured inrp1640 medium supplemented with heatactivated fetalbovine serum gibco gaithersburg md and penicillinstreptomycin gibco at —¦c with co2abl1 protein phosphatase catalyticsubunit alphappp3ca and tgf1 knockdown kd lentiviruses weregenerated using pfugwgfprnai vector by insertingshabl1 shppp3ca and shtgf1 sequence empty pfugwgfp vector was used as vector control shctrl in crc cells or ncmice the rnai sequence of abl1 ppp3ca and tgf1 were²cgttctatatcatcactga3² ²atatacgcgttctgaatactt3² ²gattatcga catggagctg3² respectivelysw480 and hct116 cells were plated in a 24wellplate andincubated at —¦c with co2 for h a multiplicity ofinfection of was added to infect sw480 and hct116 cellsovernight the infection medium was then replaced with normalcomplete growth medium cells without infection were used ascorresponding controlsproliferation and colony formation assayproliferation rate was determined using bromodeoxyuridinebrdu cell proliferation elisa kit abcam boston ma theoptical density of each sample was measured at nm using asynergy h1 microplate reader biotek winooski vtfor the clonogenic assay sw480 and hct116 cells wereplated onto 6wellplate and incubated in culture medium for days the cells were then fixed with pfa and stained with crystal violet sigmaaldrich st louis mo for h at roomtemperature the total number of colonies was counted wheneach clone contained more than cells size “ mmflow cytometric analysisfor cell cycle analysis cells were fixed in pfa for minat —¦c and treated with propidium iodide pi µgmlsigmaaldrich at room temperature for min in dark atotal of cells were analyzed by flow cytometry using abd facscalibur system becton“dickinson el paso tx thedistribution of cell cycle phases was estimated using modfitlt in mac v30 software apoptosis was further determinedby annexin v fitcconjugated thermo fischer scientificmiami ok and pi staining cells were immediately counted byflow cytometrydna extraction and sequencingfortyeight specimens of colorectal cancer tissue with clinicalliver metastases were collected dna was isolated using allprep dna ffpe kit qiagen germantown md genomic dnawas extracted by fully automated purification using promegamaxwell promega madison wi the dna concentrationwas measured fluorimetrically using the qubit dna highsensitivity kit thermo fisher scientific waltham ma anion torrent semiconductor chip sequencer was used to sequencecommon gene mutations in the tumorsin vivo studybalbc nude mice female aged weeks were purchased fromshanghai ling chang biological technology co ltd shanghaichina the mice were housed in spflevel laboratories with freeaccess to food and water and accommodated for week prior toany experiments the animal study was performed in accordancewith iacuc guidelines shabl1hct116 kd or shctrlhct nc cells — µl were subcutaneously injected tothe left flank of the mice at day posttransplantation micewere sacrificed and tumors were excised and weighed the tumorfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancervolume was calculated using digital calipers with the followingformula tumor volume volume length width22ingenuity pathway analysisto elucidate the role and action mechanism of abl1 incrc after abl1 kd high throughput realtime pcr arraywas performed by shanghai genechem co ltd shanghaichina and the data were analyzed using ingenuity pathwayanalysis ipa software to elucidate the aï¬ected molecules andsignal pathwaysimmunohistochemistryfor crc 180point tissue microarray hcolade180sur07which contained crc tissuesfrom patients and thecorresponding adjacent tissues table s4 was purchased fromshanghai outdo biotech co ltd shanghai china briefly thetissue microarray block was constructed by embedding a singletissue core mm in diameter was taken from each region informalxed paraffinembedded crc or adjacent tissue blockusing a tissue microarrayer beecher instruments silver springmd usa and was set to a blank recipient block predrilled with mm holesthe tissue microarray blocks and paraffinembedded tumorsections were cut into 7µm sections for immunohistochemicalihc analysis slides were deparaffinized and rehydrated aspreviously described followed by antigen retrievalincitrate buï¬er mm citric acid tween ph for min in —¦c water bath after washing with pbsslides were incubated with pbst with bovine serumalbumin sigmaaldrich for h slides were then incubatedovernight at —¦c with antiabl1 antibody ab15130abcam cambridge ma and developed using mouse andrabbit specific hrpdab detection ihc kit ab64264 abcamfollowing the manufacturer™s instructionsthe selection of cutoï¬ value to dichotomize the expressionlevels of abl1 was based on previously reported method []briefly the high expression level of abl1 was defined from twocriteria dab staining showed equal or darker color comparedto positive control the population of abl1positive cells washigher than all cases were independently evaluated anddiagnosed by two senior pathologists y m and l y who wereblinded to the pathologic diagnosis cases with any disagreementwere reviewed simultaneously by the original two pathologistsand a senior pathologist j w until they reach a consensuswestern blotthe western blotting assay was performed by wellestablishedprotocols as previously described primary antibodiesused in this study were antiabl1 antibody ab85947abcam cambridge ma antibcl2 antibody bcl10c4biolegend san diego ca antibclxl antibody sc santa cruz biotechnology dallas tx antibaxantibody 2d2 biolegend antiactin antibody 2f11 biolegend antigapdh antibody ff26af9biolegend antip27 antibody sc56338 santa cruzbiotechnology anticyclind1 antibody sc8396 santacruz biotechnology antiirs1 antibody ab52167abcam antiakt2 antibody ab175354 abcam antippp3ca antibody ab52761 abcam antitgf1antibody ab92486 abcam antimap2k2 antibody sc81473 santa cruz biotechnology antipi3kp11aantibody ab151549 abcam secondary antibodiesused were antimouse igg hrpconjugated secondary antibody sc516102 santa cruz biotechnology and antirabbitigg hrpconjugated secondary antibody sc2357 santacruz biotechnologyreverse transcriptionpolymerase chainreactionthe mrna level was measured using realtime polymerasechain reaction briefly total rna was extracted from culturedcells using trizol reagent thermo fisher scientific andcdna synthesis was performed using the quantitect reversetranscription kit qiagen the primers used were as followsabl1andantisense ²acaccctcccttcgtatctcag3² gadphsense ²tgacttcaacagcgacaccca3² antisense ²caccctgttgctgtagccaaa3² the realtime pcr wascarried out by using rt2 sybr rcid13 green qpcr mastermixesqiagen according to the manufacturer™s instructions all pcrswere performed in triplicate 01 01ct method was used to calculatethe relative expression levels²catcacgccagtcaacagtct3²sensestatistical analysisstatistical analyses were performed using spss spss incchicago il χ 2test was used to investigate the possiblerelationships between abl1 expression and clinic pathologicalcharacteristics mann“whitney utest was used to compare thediï¬erence in abl1 protein expression between paired coloncancer and adjacent normal colon tissues survival analysiswas performed using the kaplan“meier curve and the logranktest the values are expressed as mean ± sd comparisonsbetween two groups were conducted using student™s ttest allexperiments were carried out in triplicate results with p were considered statistically significantresultshighly expressed abl1 in crc tissue isassociated with poor clinical outcometo verify the existence of abl1 in crc tissues we comparedcrc tissues and their adjacent noncancerous tissues by ihcstaining our results showed that the immunostaining of abl1was significantly higher in crc tissues compared with adjacentnormal colon tissues p figures 1a“d table ourwestern blot and realtime pcr results confirmed the muchhigher expression level of abl1 in crc tissues compared tonormal tissues figures 1ef similarly the expression of abl1was significantly increased in diï¬erent crc cell lines comparedwith that in the normal colon cell line ncm460 figures 1ghremarkably the expression of abl1 was significantly p increased in the advanced stages stage iiiiiiv of crccompared with the early stages stage i and noncancerousfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 is highly expressed colorectal cancer tissue and cell lines a“d representative ihc staining of abl1 in normal ab and crc cd tissuesac x bd x e western blot analysis of abl1 expression in crc tumor t and adjacent normal n tissues f realtime pcr analysis of abl1expression in crc tumor crc and adjacent normal n tissues p g western blot analysis of abl1 expression in crc cell lines h realtime pcranalysis of abl1 expression in crc cell lines relative expression was normalized to ncm460 cells p i realtime pcr analysis of abl1 expression incrc tumors at different clinical stages relative expression was normalized to adjacent normal n tissues p table expression of abl1 in colon cancer and adjacent tissuestable c1222c deletion in exon of abl1variablesnoexpression levelspvariablesexpression levelsor95 cipneglowhighno no mutation mutationnormalcolon cancergenderfemaleadjacent normal colon tissues neg negativetissues figure 1i with a median followup of monthsranging from to months our survival analysis showed thatthe patients with high abl1 expression death had a lowersurvival rate compared to patients with low abl1 expression death p figure s5 these results suggested thatabl1 is a potential oncogene abl1 and that its expression waspositively associated with the clinical stage in patients with crcc1222c deletion in exon of abl1 inrelation to the tnm stageprevious studies have shown the mutations of the abl1 geneare of major clinical relevance to study the possiblemutations in patients with crc we performed dna sequencingour results indicated that a mutation of abl1 was presentin males and females of patients with crc females and males this mutation occurred in exon ofthe abl1 gene and all mutations were found to be deletion““ maletnm stage““ofthe c1222c nucleotide sequence within this exon theincidence rate of mutation was in females and malestable figure additionally the analysis of patients withmutations and the corresponding stages revealed that patientswere in stages “ and in stages “ the tnm stage was asignificant risk factor for c1222c deletion the results suggestedthat c1222c deletion is involved in crc carcinogenesisinterference of abl1 decreased theproliferation and enhanced the apoptosisof sw480 and hct116 cellsto further investigate the role of abl1 in crc carcinogenesiswe used lentivirus vector to downregulate abl1 expression insw480 and hct116 cells after infection both cell lines showedfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure structural domain and c1222c deletion in exon of the abl1 genefigure depletion of abl1 decreases the proliferation of crc cells a representative fluorescent images showing gfppositive cells after infection images weretaken under x b western blot detects the abl1 expression in cells transfected infected with shctrl or shabl1 c representative images of colony formationassay using crc cells infected with shctrl or shabl1 cells without infection was used as control con d quantification of colony numbers data are shown asmean ± sd p compared with control e brdu assay detects the proliferation of sw480 and hct116 cell lines infected with shctrl or shabl1 cellswithout infection was used as control con p compared with control“ of average gfppositive rate figure 3a our westernblot results showed a significant decrease of abl1 protein levelfigure 3b indicating a successful downregulation after rnainterference to evaluate the proliferation of crc cells afterabl1 depletion we performed a clonogenic assay figure 3ccompared with the control group the number of clones inthe shabl1 group was obviously decreased figure 3d ourbrdu proliferation assay confirmed that the proliferation offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 causes cell cycle arrest and apoptosis in sw480 and hct116 cells ab representative cell cycle analysis of sw480 a andhct116 b infected with shctrl or shabl1 cells without infection was used as control con c quantification of cell cycle distribution in g1 s g2m phases p p compared to control group d western blot analysis of p27 and cyclin d1 expressions in crc cells infected with shctrl or shabl1 cellswithout infection was used as control configure depletion of abl1 increases the apoptosis of crc cells flow cytometry detected apoptosis of sw480 a and hct116 b cells infected with shctrl orshabl1 cells without infection was used as control con c quantification of apoptotic cells data are shown as mean ± sd p compared with controld western blot analysis of bax bclxl and bcl2 expression in crc cells infected with shctrl or shabl1 cells without infection was used as control conshabl1 cells was obviously reduced compared with that ofthe control cells figure 3e additionally our flow cytometryresults showed more cells were arrested in s phase afterabl1 depletion while fewer cells in g2m phases were foundfigures 4a“c indicating downregulation of abl1 inhibitedcell cycle progression of crc cells to validate these data wedetected p27 a negative regulator of cell cycle progression andfound its expression was significantly increased in cells infectedwith shabl1 vector figure 4d on the contrary cyclin d1 wasdecreased in abl1depleted crc cells figure 4dnext we performed flow cytometric analysis to examinethe apoptosis of abl1depleted crc cells figures 5ab wefrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 inhibits crc tumor growth in vivo a representative image of mice injected with hct116 cells infected with shctrl nc or shabl1kd b representative tumor images showing depletion of abl1 decreased the size of crc tumors body weight c tumor volume d and tumor weight e weremeasured at day after inoculation p compared to nc groupfound downregulation of abl1 significantly increased apoptosisin crc cells as compared with the control group p figure 5c the expression of the apoptosisrelated proteinbcl2associated x bax was obviously increased while bcelllymphomaextralarge bclxl and bcell lymphoma bcl2were remarkably decreased in the shabl1 group figure 5dtaken together these results suggest depletion of abl1 increasesthe apoptosis of crc cellsdepletion of abl1 inhibited crc tumrowth in vivoin order to examine the involvement of abl1 in regulatingcrc tumor growth we inoculated hct116 cells infected withshctrl nc group or shabl1 kd group into balbc nudemice figure 6a as shown in figure 6b the tumor growth wasremarkably inhibited in the kd group compared with the ncgroup interestingly we also observed a significant bodyweightincrease in the kd group figure 6c as compared to controlabl1 depletion caused a significant reduction in tumor volumefigure 6d the average tumor weight in kd xenografts wasobviously lower than that in the nc group ± mgvs ± mg p figure 6e taken together ouranimal experiments demonstrated that abl1 knockdown couldinhibit crc tumor growth in vivoabl1 interference inhibited tgf1 via thepi3kaktirs1 pathway and ppp3cato elucidate the molecular pathways regulated by abl1 in crcwe performed high throughput pcr array from xenografts inabl1 kd or nc mice our ipa results identified upregulatedgenes and downregulated genes in xenografts from kdmice compared with those from nc mice further analysisrevealed that these diï¬erentially expressed genes were involvedin multiple biological functions and pathogenesis of multiplediseases figure s1as shown in figures s2 s3 and tables s1“s3 the tgfand pi3kakt pathways were inhibited by depletion of abl1the associated molecules of the two pathways including tlr4akt2 il4r camk2d ppp3cb map2k2 pdia3 irs1itpr3 abl1 atf4 ppp3ca ca2 cpt1a csrp1 ctsv fn1lamp2 ptgs2 runx2 s100a4 and spp1 were mapped withabl1 gene to show a predicted interaction network figure 7ato verify this interaction we examined the levels of proteins intgf and pi3kakt pathways in xenografts of nc and kd micefigure 7b our western blot results showed that knockdown offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 interacts with pi3kakt and tgf1 pathways a molecule network generated by ipa showing interactions among abl1 pi3kakt and tgf1pathways up and downregulated genes are shown in red and green respectively b western blot analysis of predicted interactive proteins in xenografts fromabl1 knockdown kd or control nc mice the gene of œ is detected by multiple probes and it is statistically significantabl1 significantly decreased irs1 akt2 ppp3ca and tgf1 expression while did not change the expression of map2k2compared with those in the nc groupto further verify the involvement of tgf1 in the regulationof pi3kakt pathway we generated a tgf1depletion hct cell line by lentivirus infection figure 8a our western blotresult showed that the expression of tgf1 was significantlydownregulated after infection figure 8b as expected thekey proteins in pi3kakt pathways were deactivated upontgf1depletion figure 8c including irs1 phosphopi3kand akt according to the findings in the previous study thedownregulated gene ppp3ca found in abl1 kd mouse isinvolved in the regulation of the pi3kakt pathway wenext investigated the interaction between ppp3ca and abl1by establishing a ppp3ca knockdown cell line figures 8dewe found the depletion of ppp3ca significantly decreasedthe expression of abl1 figure 8f to validate the regulatoryrole of ppp3ca we also examined the abl1 expression inppp3ca overexpressed cells and found the expression of abl1was elevated by upregulated ppp3ca figure s4 indicatingppp3ca is a positive regulator of abl1discussionas a ubiquitously expressed nonreceptor tyrosine kinase abl1has been reported to be associated with glioblastoma and breastcancer in this study we examined the role of abl1 incrc progression the results indicated that abl1 might play animportant role in crc which is associated with the mutation andexpression of the abl1 genewe found the expression of abl1 was remarkably elevatedin crc tissues and cell lines figure which is correspondedto the survival rate among patients with crc figure s5indicating abl1 is a potential oncogene in crc “moreover the mutation of abl1 was also elevated in crcpatients based on the results from previous studies the mutationrate of the abl1 gene is relatively higher in men than in womenpatients with crc worldwide in a previous study theabl1 gene was found to be mutated in of patients withcrc at sir ganga ram hospital delhi india in the presentstudy the mutation rate was much higher in crcpatients accepted in our hospital the diï¬erent races of patientsfrom diï¬erent regions of the world reported in the two studiesfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure tgf1 knockdown deactivates pi3kakt pathway in hct116 cells a representative images of hct116 cells infected with gfpcontaining lentivirusexpressing shrna against tgf1 gfp positive lentivirus with scramble shrna was used as control images were captured under 200x magnification bknockdown of tgf1 was confirm by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading control cwestern blot examined irs1pi3kakt pathway in hct116 cells after knockdown of tgf1 d western blot examined irs1pi3kakt pathway in hct116 cellsinfected with gfpcontaining lentivirus expressing shrna against ppp3ca gfp positive lentivirus with scramble shrna was used as control e representativefluorescent images of hct116 cells at h postinfection images were captured under 200x magnification f expression of abl1 after ppp3ca knockdown wasdetermined by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading controlcould be a possible reason causes the diï¬erence of abl1 mutationrates “ gene mutations are often involved in tumorigenesisthe clustered deletions were found in abl1 notch1 retstk11 gna11 and jak3 genes in crc melanoma and nonsmall cell lung cancers additionally the abl1 mutationdata in tcga showed that an average mutation rate of abl1is in coad patients the high mutation rate isconsistent with the findings in this study that abl1 mutationcorrelates with the oncogenesis of crc to the best of ourknowledge the present study presents a novel mutation inexon in which c1222c deletion occurred this deletion wasrelatively higher in female patients than in male patients thehigher distribution of this deletion at the higher tnm stage inpatients with crc suggests that this deletion might be relatedto tumorigenesis of crc however further investigation withlarger sample size is needed to elucidate the relationship andmechanism between c1222c deletion and crc progressionto determine the role of abl1 in crc progression wedownregualted abl1 expression in crc cell lines and foundthat the cell cycle was arrested at s phase figure theseobservations are consistent with previous reports thatthenumber of cells in s phase was increased when abl1 wasinhibited by imatinib or sti571 in u2os hela and a549 cells it is wellreported that p27 inhibits g1s transition ofthe cell cycle while cyclin d1 is a key regulator of cell entryinto the s phase allowing cells to enter the s phase smoothlyfrom the g1 phase our study provides direct evidencethat abl1 interference increased p27 expression and decreased incyclin d1 expression figure which is similar to the increasedp27 expression and decreased cyclin d1 expression found incells treated with nilotinib an abl1specific inhibitor however previous studies showed that when abl1 expressionwas inhibited by nilotinib the number of cells in the g0g1 phasewas increased while the number of cells in s and g2m phases wasdecreased which is contradictory to the finding in this studythat downregulation of abl1 arrested crc cells at s phase thismight be due to the varied function of abl1 in diï¬erent tissuesand cell types abl1 controls cell apoptosis via downstream moleculessuch as puma bax and p73 as well as by changingfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancermembrane potential depletion of abl1 inducedapoptosis of crc cells observed in this study is consistentwith the findings ofthese studies studies have reportedthat abl inhibitor danusertib treatment significantly decreasedthe expression of bclxl and bcl2 while increasing theexpression of bax similarly we found increasedbax expression and decreased levels of bcl2 and bclxl after downregulation of abl1 in crc cells figure indicating abl1 is involved in the regulation of apoptosis incrc cellsinvolved in cell proliferation migrationfurthermore information obtained using the ipa indicatedthe expressions of numerousthat after abl1 knockdowngenesinvasiondiï¬erentiation death and survival were aï¬ected figures s1“s3 tables s1“s3 the results demonstrated that abl1 mightplay a pivotal role in crc progression especiallythe tgf and pi3kakt pathways were inhibited afterabl1 interferenceit has been reported that abl1 regulates tgf signaling which is associated with tumor progression by modulatingangiogenesis in crc resulting in poor prognostic outcome“ studies have demonstrated thattreatment withan abl1 inhibitor significantly reduced the tgf level similarly we found the expression of tgf1 wassignificantly inhibited after abl1depletion figure thisindicated that abl1 is a positive regulator of tgf signalpathways as one of the tgfaï¬ected downstream signals thepi3kakt pathway plays a crucial role in tumorigenesisitassociated withproliferation apoptosisinvasion and metastasis of cancercells insulin receptor substrates irs including irs1and irs1 are a downstream messenger of the pi3k pathway our study provides novel evidence that abl1 mightinteract with tgf1 via pi3kaktirs1 that is involved incrc progressionexpression of proteinsregulatestheca2aandcalcineurincalmodulindependentserinethreonine protein phosphatase has been reported topromote intestinal tumor development and crc tumorigenesis the expression of calcineurin a specifically increases inhuman crc cell lines in the present study we foundthat ppp3ca which is also known as an alpha isoform ofthe calcineurin catalytic subunit was inhibited afterknockdown of abl1 figure 7b this finding provides novelevidence that abl1 might interact with the ppp3ca oncogenein crc carcinogenesisconclusionin conclusion we found a high level of abl1 expression incrc tissue and cells which was associated with the tnmstages a novel mutation of c1222c deletion in exon of theabl1 gene was found and was associated with the crc stagedepletion of abl1 decreased the growth of crc cell lines bothin vitro and in vivo by inhibiting tgf pathway these resultsdemonstrated novel understandings of the function of abl1during the progression of crc thus provides a clinically viablestrategy for crc therapydata availability statementthe raw data supporting the conclusions of this will bemade available by the authors without undue reservationethics statementthe studies involving human participants were reviewed andapproved by ethics committee of shaanxi people™s hospital thepatientsparticipants provided their written informed consent toparticipate in this study the animal study was reviewed andapproved by ethics committee of shaanxi people™s hospitalauthor contributionsall authors have a significant scientific contribution to all aspectsof this studyfundingstudy wasthissupported by national natural sciencefoundation of china”youth projects grant no shaanxi natural science foundation grant nos 2015jq8321and 2019jm547 shaanxi innovative talents cultivate programgrant no 2017kct28 and operating expenses of basicscientific research project of xi™an jiaotong university grantno xzy012019112supplementary materialthe supplementary materialonline202001385fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences ouerhani s bougatef k soltani i elgaaied ab abbes s menif s theprevalence and prognostic significance of kras mutation in bladder cancerchronic myeloid leukemia and colorectal cancer mol biol rep “ 101007s1103301325128 brenner h kloor m pox cp colorectal cancer lancet “ 101016s0140673613616499 navarro m nicolas a ferrandez a lanas a colorectal cancer populationscreening programs worldwide in an update world j gastroenterol “ 103748wjgv23i203632 favoriti p carbone g greco m pirozzi f pirozzi re corcione fworldwide burden of colorectal cancer a review updates surg “ 101007s133040160359yjauhri m bhatnagar a gupta s shokeen y minhas s aggarwal stargeted molecular profiling of rare genetic alterations in colorectalcancer 101007s1203201608202sequencing med oncolnextgenerationusing peng x luo z kang q deng d wang q peng h foxq1mediates the crosstalk between tgfbeta and wnt signaling pathways inthe progression of colorectal cancer cancer biol ther “ frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancer rossner f gieseler c morkel m royer hd rivera m blaker h uncoupling of egfrras signaling and nuclear localization of ybx1in colorectal cancer oncogenesis 5e187 101038oncsis alpay k farshchian m tuomelainhibition ofsiljamaki ecancer9e105526 101371 pone0105526cells highly sensitivealetj sandholm j aittokallio krenderscabl kinaseactivityto mitoxantrone plos one tian xq guo ff sun df wang yc yang l chen sl downregulationof znf278 arrests the cell cycle and decreases the proliferation of colorectalcancer cells via inhibition of the erkmapk pathway oncol rep “ 103892or20176031 udden sm moritafujimura y satake m ikawa s cabl tyrosinefate “kinase modulates p53dependent p21 induction and ensuing celldecision in response to dna damage cell signal 101016jcellsig201310005 cai s cheng x liu y lin z zeng w 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Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm

" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patient™s quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage ii“iii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately “ of patients with crc were diagnosed with acute colonic obstruction [“] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [“] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [“] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatient™s symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage ii“iiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudent™s ttest and wilcoxon test for continuous variables and the χ2 and fisher™s exact tests for categoricalvariables were conducted survival curves were generated using the kaplan“meier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range “ “genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n “ “ a clavien“dindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ‰¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patient™ssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency “ “duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml“ ““ “ before afterstoma creation “morbidity30day complication cda iii anastomosticleakagehospital stayaclavien“dindo classificationmedian range days “ “primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplan“meier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n “ bts n “ p “survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [“] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ ˆ’hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ overall suvivales vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ without obstruction [“] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment [“]the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ hr ± hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage ii“iii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors™ contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number “consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin “ pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg “ pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer 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" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen people™s hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi people™s hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudeck™s critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudeck™s point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patient™s right side and the first assistant stood atthe patient™s left foot side the laparoscopic monitor wasplaced on the patient™s right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks™ white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldt™s fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately “ cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately “ cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately “ cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about “cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about “ cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a χ2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades “ and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willian™s report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm “ cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm “ cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of “ cm in the author™s medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages i“iii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed “ weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of “yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side “ cm above thetumor according to the location ofthebranches of the severed vessels are determined and “cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors™ confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon™ s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors™ contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi people™s hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first people™shospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen people™s hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg “ macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet “lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol “ williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients™ survival br j durg “ pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg “ wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes™ c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg “ monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum “law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg “kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol “kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institution™s experience with patients with rectalcancer j korean soc coloproctol “ zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg “kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg “ poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol “ yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum “seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg “lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery “zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg “sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr “van tonder jj boon jm becker jhr anatomical considerations onsudeck™s critical point and its relevance to colorectal surgery clin anat“cirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c"

" Apoptosis of NSCLC A549 cells after ionizing radiation (IR). Apoptosis in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) IR (8.0?Gy) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). Suppression of ionizing radiation-induced phosphorylated-Akt (p-Akt) upregulation by anti-miR-21 in NSCLC A549 cells. The expression level of the phosphorylated or total Akt in A549 cells transfected with anti-miR-21 or anti-miR-NC for 48?h followed by ionizing radiation (8?Gy) with or without 10?ng/mL PI3K constituent activator IGF-1 was measured by Western blot. Representative of three independent experiments was shown. Knockdown of miR-21 promoted NSCLC A549 cells apoptosis via inactivation of PI3K-Akt pathway. Apoptosis induced by 8?Gy ionizing radiation in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) PI3K activator IGF-1 treatment (10?ng/mL) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). 1306016 3069 Clin Radiol Clin Radiol Clinical radiology 0009-9260 1365-229X 24857677 4105980 10.1016/j.crad.2014.03.020 NIHMS587373 Revisiting the relationship between tumour volume and diameter in advanced NSCLC patients: An exercise to maximize the utility of each measure to assess response to therapy Nishino M. a * Jackman D.M. b DiPiro P.J. a Hatabu H. a Jnne P.A. b Johnson B.E. b aDepartment of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. 75 Francis St. Boston MA 02215 USA bDepartment of Medical Oncology and Department of Medicine Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA *Guarantor and correspondent: M. Nishino Department of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA. Tel.: +1 617 582 7163; fax: +1 617 582 8574. Mizuki_Nishinodfci.harvard.edu (M. Nishino) 30 5 2014 22 5 2014 8 2014 01 8 2015 69 8 841 848 2014 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. 2014 AIM To revisit the presumed relationship between tumour diameter and volume in advanced non-small-cell lung cancer (NSCLC) patients and determine whether the measured volume using volume-analysis software and its proportional changes during therapy matches with the calculated volume obtained from the presumed relationship and results in concordant response assessment. MATERIALS AND METHODS Twenty-three patients with stage IIIB/IV NSCLC with a total of 53 measurable lung lesions treated in a phase II trial of erlotinib were studied with institutional review board approval. Tumour volume and diameter were measured at baseline and at the first follow-up computed tomography (CT) examination using volume-analysis software. Using the measured diameter (2r) and the equation calculated volume was obtained as (4/3) ?r3 at baseline and at the follow-up. Percent volume change was obtained by comparing to baseline for measured and calculated volumes and response assessment was assigned. RESULTS The measured volume was significantly smaller than the calculated volume at baseline (median 11488.9 mm3 versus 17148.6 mm3; p < 0.0001) with a concordance correlation coefficient (CCC) of 0.7022. At follow-up the measured volume was once again significantly smaller than the calculated volume (median 6573.5 mm3 versus 9198.1 mm3; p = 0.0022) with a CCC of 0.7408. Response assessment by calculated versus measured volume changes had only moderate agreement (weighted ? = 0.545) with discordant assessment results in 20% (8/40) of lesions. Calculated volume based on the presumed relationship significantly differed from the measured volume in advanced NSCLC patients with only moderate concordance in response assessment indicating the limitations of presumed relationship. CMAJ CMAJ 9711805 CMAJ : Canadian Medical Association Journal 0820-3946 1488-2329 Canadian Medical Association 24638027 4016095 10.1503/cmaj.131385 186e296 Practice Five Things to Know About... Screening for lung cancer Kennedy Sean Baerlocher Mark Otto MD School of Medicine (Kennedy) McMaster University Hamilton Ont.; Department of Radiology (Baerlocher) Royal Victoria Hospital Barrie Ont. Correspondence to: Sean Kennedy sean.kennedymedportal.ca 13 5 2014 13 5 2015 186 8 E296 E296 1995-2014 Canadian Medical Association 2014 0042124 4284 Int J Cancer Int. J. Cancer International journal of cancer. Journal international du cancer 0020-7136 1097-0215 24806617 4200482 10.1002/ijc.28958 NIHMS594253 p38 MAPK inhibits breast cancer metastasis through regulation of stromal expansion Hong Bangxing 1 Li Haiyan 1 Zhang Mingjun 1 Xu Jingda 2 Lu Yong 1 Zheng Yuhuan 1 Qian Jianfei 1 Chang Jeffrey T 3 Yang Jing 2 Yi Qing 1 1Department of Cancer Biology Lerner Research Institute Cleveland Clinic Cleveland OH USA 2Department of Lymphoma and Myeloma University of Texas MD Anderson Cancer Center Houston TX USA 3Department of Integrative Biology & Pharmacology University of Texas at Houston Houston TX USA Corresponding author: Qing Yi MD PhD Department of Cancer Biology Cleveland Clinic Lerner Research Institute 9500 Euclid Avenue/NB40 Cleveland OH 44195 USA. Tel: 216-636-7532; Fax: 216-444-3164; yiqccf. 31 5 2014 16 5 2014 1 1 2015 01 1 2016 136 1 34 43 p38 MAPK signaling controls cell growth proliferation and the cell cycle under stress conditions. However the function of p38 activation in tumor metastasis is still not well understood. We report that p38 activation in breast cancer cells inhibits tumor metastasis but does not substantially modulate primary tumor growth. Stable p38 knockdown in breast cancer cells suppressed NF-?B p65 activation inhibiting miR-365 expression and resulting in increased IL-6 secretion. The inhibitory effect of p38 signaling on metastasis was mediated by suppression of mesenchymal stem cell (MSC) migration to the primary tumor and sites of metastasis where MSCs can differentiate into cancer-associated fibroblasts to promote tumor metastasis. The migration of MSCs to these sites relies on CXCR4-SDF1 signaling in the tumor microenvironment. Analysis of human primary and metastatic breast cancer tumors showed that p38 activation was inversely associated with IL-6 and vimentin expression. This study suggests that combination analysis of p38 MAPK and IL-6 signaling in patients with breast cancer may improve prognosis and treatment of metastatic breast cancer. p38 MAPK Breast cancer Metastasis microRNA IL-6 J Mol Diagn J Mol Diagn The Journal of Molecular Diagnostics : JMD 1525-1578 1943-7811 American Society for Investigative Pathology 24813172 4078366 S1525-1578(14)00074-9 10.1016/j.jmoldx.2014.03.006 Regular Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing Abel Haley J. ? Al-Kateb Hussam   Cottrell Catherine E.   Bredemeyer Andrew J.   Pritchard Colin C. ¡ Grossmann Allie H. § Wallander Michelle L. ¶ Pfeifer John D.   Lockwood Christina M.   Duncavage Eric J. eduncavagepath.wustl.edu   ? ?Department of Genetics Washington University St. Louis Missouri  Department of Pathology and Immunology Washington University St. Louis Missouri ¡Department of Laboratory Medicine University of Washington Seattle Washington §Department of Pathology University of Utah and ARUP Laboratories Salt Lake City Utah ¶ARUP Institute for Clinical and Experimental Pathology Salt Lake City Utah ?Address correspondence to Eric J. Duncavage M.D. Department of Pathology and Immunology Division of Anatomic and Molecular Pathology Division of Laboratory and Genomic Medicine 660 Euclid Ave. #8118 St. Louis MO 63110. eduncavagepath.wustl.edu 1 7 2015 7 2014 16 4 405 417 6 3 2014 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. 2014 American Society for Investigative Pathology and the Association for Molecular Pathology This document may be redistributed and reused subject to certain conditions. The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers six KMT2A rearranged leukemias and 77 ALK/KMT2A rearrangement“negative cancers previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools including Breakdancer ClusterFAST CREST and Hydra. Using Breakdancer and ClusterFAST we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases no false-positive identifications were made by Breakdancer or ClusterFAST. Further we identified one ALK rearranged case with a noncanonical intron 16 breakpoint which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel“based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing. Biomed Res Int Biomed Res Int BMRI BioMed Research International 2314-6133 2314-6141 Hindawi Publishing Corporation 24524077 3913339 10.1155/2014/485067 Research Investigating the Feasibility of Rapid MRI for Image-Guided Motion Management in Lung Cancer Radiotherapy http://orcid./0000-0002-3275-4160 Sawant Amit 1 * Keall Paul 2 Pauly Kim Butts 3 Alley Marcus 3 Vasanawala Shreyas 3 Loo Jr. Billy W. 3 Hinkle Jacob 4 Joshi Sarang 4 1University of Texas Southwestern Medical Center Dallas TX 75235 USA 2University of Sydney Sydney NSW 2006 Australia 3Stanford University Stanford CA 95305 USA 4University of Utah Salt Lake City UT 84112 USA *Amit Sawant: amit.sawantutsouthwestern.edu Academic Editor: Jack Yang 2014 12 1 2014 2014 485067 17 4 2013 6 11 2013 7 11 2013 Copyright 2014 Amit Sawant et al. 2014 This is an open access distributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Cycle-to-cycle variations in respiratory motion can cause significant geometric and dosimetric errors in the administration of lung cancer radiation therapy. A common limitation of the current strategies for motion management is that they assume a constant reproducible respiratory cycle. In this work we investigate the feasibility of using rapid MRI for providing long-term imaging of the thorax in order to better capture cycle-to-cycle variations. Two nonsmall-cell lung cancer patients were imaged (free-breathing no extrinsic contrast and 1.5?T scanner). A balanced steady-state-free-precession (b-SSFP) sequence was used to acquire cine-2D and cine-3D (4D) images. In the case of Patient 1 (right midlobe lesion ~40?mm diameter) tumor motion was well correlated with diaphragmatic motion. In the case of Patient 2 (left upper-lobe lesion ~60?mm diameter) tumor motion was poorly correlated with diaphragmatic motion. Furthermore the motion of the tumor centroid was poorly correlated with the motion of individual points on the tumor boundary indicating significant rotation and/or deformation. These studies indicate that image quality and acquisition speed of cine-2D MRI were adequate for motion monitoring. However significant improvements are required to achieve comparable speeds for truly 4D MRI. Despite several challenges rapid MRI offers a feasible and attractive tool for noninvasive long-term motion monitoring. 1. Introduction Respiratory motion causes significant uncertainties in tumor delineation radiotherapy (RT) dose calculations and delivery particularly in the case of thoracic tumors (e.g. lung liver) [1]. The management of respiratory motion has been an active area of research over the last decade. Several investigational as well as clinically implemented respiratory motion management strategies have been described in the literature [1]. However a common limitation of most of these strategies is that they rely on image-guidance techniques that make simplifying assumptions about respiratory motion and do not adequately capture cycle-to-cycle variations which invariably occur in all patients. Modern motion-managed radiotherapy typically uses four-dimensional computed tomography (4DCT) as the tool of choice for pretreatment anatomic imaging (also termed as œCT simulation or œCT-sim in the literature). In this technique CT projections are acquired over several respiratory cycles from successive œslabs in the body. At the same time an external surrogate (e.g. an optical marker) records the amplitude of respiration. Based on the surrogate motion trace the reconstructed slices are sorted into 6“10 volumes over a single respiratory average cycle where each volume represents a specific phase of respiration (inhalation through exhalation) [2“4]. This retrospectively reconstructed œmovie of a single respiratory cycle serves as the anatomical ground truth for all subsequent stages of radiotherapy (contouring treatment planning and dose delivery). It is well recognized however that respiratory motion is far more complex than can be characterized by a single average cycle. Cycle-to-cycle variations such as baseline shifts and changes in the amplitude and/or frequency of the respiratory waveform are inadequately accounted for in 4DCT-based planning and can lead to significant geometric and therefore dosimetric errors [5]. Furthermore binning CT projection data acquired over several cycles into a single cycle leads to severe image artifacts. For example Yamamoto et al. found that 45 of 50 patients had at least one artifact with mean magnitude of 11.6?mm (range: 4.4“56.0?mm) [6]. In a separate study Persson et al. found that 4DCT artifacts caused significant uncertainties in the delineation of the gross tumor volume (GTV) in 16 out of 19 patients [7]. Finally the equivalent dose for 4DCT is quite high (29“40?mSv) about 4 times higher than that for 3DCT (3“10?mSv) [8]. Such high imaging dose discourages long-term monitoring and frequent imaging. Due to these limitations 4DCT-based image guidance provides an incomplete picture of respiration-induced spatial and temporal changes in the thoracic anatomy. The aim of this work is to investigate the feasibility of using rapid magnetic resonance imaging (MRI) as a nonionizing imaging modality to capture long-term and/or frequent information about respiratory motion and its effects on the movement and deformation of lung tumors and surrounding critical ans. The fundamental difference and therefore advantage of cine MRI are that unlike 4DCT the MR image (i.e. slice or volume) is acquired prospectively thereby capturing an actual instance of the patient anatomy which is closer to reality compared to an average estimate of the anatomical state that is represented by 4DCT. Prospective acquisition also enables MRI to overcome the two main challenges that limit the utility of 4DCT images namely the ability to capture cycle-to-cycle variations and elimination of binning-related image artifacts. In addition due to the fact that MRI does not involve ionizing radiation there is no dose penalty for repeated imaging (as opposed to 4DCT). The use of rapid cine-2D as well as 4D MRI for radiotherapy guidance has been previously reported in the literature. In cine-2D MRI a slice of the anatomy is selected at arbitrary orientation and imaged repeatedly in time. 4D MRI is conceptually similar except that in this case an entire volume is selected and imaged. Plathow et al. have reported cine-2D imaging of lung cancer patients at ~3 frames per second (fps) [9] and 4D imaging of malignant pleural mesothelioma patients at ~1 volume/s [10] under slow-breathing conditions using a 1.5?T scanner. Von Siebenthal et al. have reported on a 4D MR imaging technique using retrospective stacking of cine-2D slices [11]. Biederer et al. report 4D MRI of a ventilated chest phantom that uses porcine lung with embedded agarose nodules to simulate tumors [12]. More recently Cai et al. have reported a 4D MRI study of a moving phantom using a technique that uses retrospective sorting of cine-2D slices [13]. To our knowledge there has been no systematic study of rapid lung MRI in the context of image-guided radiotherapy (IGRT) motion management under realistic (prospective acquisition free-breathing human subjects) conditions. In this work we present a pilot investigation of prospective rapid cine-2D and cine-3D (commonly termed as œ4D in radiotherapy and the MRI literature) MRI of two nonsmall-cell lung cancer (NSCLC) patients under free-breathing conditions without externally administered contrast. Subsequently we compute and analyze the motion trajectories of tumors and structures of interest. Our current goal is to demonstrate the feasibility and the utility of rapid MR imaging to monitor respiratory motion over multiple cycles and obtain guidance information about the motion deformation and the interplay between lung tumors and surrounding critical ans. Our long-term goal (beyond the current scope) is to use the information obtained from rapid MRI to augment and potentially correct 4DCT images. 2. Methods 2.1. Imaging of NSCLC Patients Two NSCLC patients were imaged following informed consent. Patient number 1 was a 67-year old female with an ~40?mm diameter right midlobe tumor. Patient number 2 was an 80-year old male with an ~60?mm diameter left upper-lobe tumor. Both patients were scanned on a 1.5?T scanner (GE Signa). Both patients were scanned in the supine position under free-breathing conditions and without externally administered contrast. For each patient a 4-channel cardiac coil was centered around the tumor. cine-2D time series in the coronal and sagittal planes were acquired using a balanced steady-state free precession (b-SSFP) sequence and the images were reconstructed using the vendor's in-built software. In all cases except one (Patient number 1 coronal series) half-Fourier acquisition was used in order to achieve higher imaging speed. In the case of Patient number 2 an additional 3D+t (4D) scan of a tumor-inclusive coronal slab (8 slices each 5?mm thick) was acquired using the b-SSFP sequence in the 3D mode and in conjunction with parallel imaging (acceleration = 4). The 4D images were reconstructed using the autocalibrating reconstruction for Cartesian imaging (ARC) algorithm [14]. Table 1 summarizes the image acquisition parameters for the cine-2D and the 4D acquisitions. 2.2. Motion Analysis For each time series from Table 1 the motion trajectories of the tumor and structures of interest were determined as follows. A fluid-flow-based deformable image registration previously validated for RT applications [15“17] was applied to each time series to compute deformation vector fields (DVFs) across the temporal dimension. In order to reduce errors and achieve high computation speed (i.e. fewer iterations) the registration was performed in two stages-rigid registration which accounted for gross translation and affine transformations of the tumor and ans followed by deformable registration which accounted mainly for tumor and an deformation. For each time series a reference image was selected (typically at mid-inhale) and ~15 points each on the tumor boundary and the diaphragm were manually selected. Subsequently the motion trajectory of each pixel on a contour was determined from the DVFs. The validity of using diaphragmatic motion as a surrogate for tumor motion was examined by calculating the correlation between the average motion trajectory of the pixels comprising the diaphragm boundary with the average trajectory of the pixels comprising the tumor boundary. The presence of complex motion such as tumor rotation and/or deformation was tested by comparing the motion trajectory of the tumor centroid with those of the selected points on the tumor boundary. 3. Results and Discussion Figure 1 shows MR images acquired from Patient number 1 (Figures 1(a) and 1(b)) and Patient number 2 (Figures 1(c) and 1(d)). The acquisition times per image ranged from ~0.15 to 0.27?s”speeds adequate for monitoring most respiratory motion. In each case the tumor mass (indicated by an arrow) can be clearly delineated against the background of lung parenchyma. Figure 2(a) shows a frame from the 4D acquisition from Patient number 2. A surface rendered tumor-inclusive volume-of-interest in four different respiratory phases is shown in Figure 2(b). Both the tumor as well as the surrounding anatomy exhibit significant deformation from phase to phase. Figure 3 shows motion trajectories extracted from two time series one from each patient. MRI-based monitoring over multiple respiratory cycles yields some interesting observations. In the case of Patient number 1 there is little cycle-to-cycle variation in the respiratory pattern as evidenced by the motion trajectory of the diaphragm. Furthermore the motion of the tumor centroid is well correlated with the motion of the diaphragm (Figure 3(a); R2 = 0.99) indicating that in this case diaphragmatic motion is an appropriate surrogate for tumor motion. Finally the motion of individual points on the tumor boundary (i.e pixels comprising the edges of the tumor mass) is well correlated with that of the tumor centroid (Figure 3(b); R2 = 0.9 to 1.0) indicating the absence of any significant rotation or deformation in the tumor mass. In the case of Patient number 2 while the respiratory pattern is quite regular (as seen from the motion trajectory of the diaphragm) the motion of the tumor centroid is very poorly correlated with diaphragmatic motion (Figure 3(c); R2 = 0.16) and shows significant cycle-to-cycle variation. This behavior indicates that in this case diaphragmatic motion is a poor surrogate for tumor motion. Furthermore the motion of the tumor centroid is also relatively poorly correlated with that of individual points on the tumor boundary (Figure 3(d); R2 = 0.56 to 0.94) indicating the occurrence of significant rotation/deformation of the tumor mass. The complex motion observed in Patient number 2 is likely due to the proximity of tumor to the cardiac wall which almost touches the edge of the tumor (Figure 1(c)) and serves as a second actuator of motion (the first being the diaphragm). These results demonstrate that the current clinical practice of using the motion of the diaphragm (or external or internal surrogates for diaphragmatic motion) has significant limitations when the tumor mass is located in the proximity of other moving structures. The goal of this work was to demonstrate the feasibility and the potential advantages of using rapid MRI as a pretreatment image-guidance tool for lung RT. These early results from rapid MRI of NSCLC patients show that for guidance-quality imaging the inherent contrast presented by the tumor mass and critical structures against the signal-poor lung parenchyma enables us to sacrifice SNR in order to achieve adequate acquisition speed to capture respiratory motion. Furthermore in the case of Patient number 2 we observe that through long-term prospective MR imaging one can capture spatiotemporal effects that are not captured by 4DCT. This is due to the fact that 4DCT projections are sorted using an external surrogate for diaphragmatic motion thereby implicitly assuming that a perfect correlation exists between diaphragmatic motion and tumor motion. The choice of a 1.5?T scanner for this work was motivated by the fact that several lung motion investigations have been performed at this field strength [12 18]. Observer studies comparing 1.5?T and 3?T scanners for lung MRI show that there is no significant difference in overall image quality [19 20] suggesting that the expected benefits of higher SNR at 3?T are somewhat mitigated due to the accompanying increase in susceptibility artifacts. Furthermore at this initial stage we chose to use existing coils and sequences. As seen from the results while this strategy was adequate for cine-2D imaging very large improvements in acquisition speed are required for truly 4D MRI. This is evidenced by the fact that even with the use of parallel acceleration = 4 the acquisition time for the 4D time series shown in Figure 2 was ~1.5?s/volume. Thus there is much room for exploration of other rapid MRI sequences and for developing sequences specifically optimized for RT guidance. In particular we expect the largest improvements in imaging speed to come from strategies based on sparse sampling and reconstruction such as k-t Broad-use Linear Acquisition Speed-up Technique (k-t BLAST) and its parallel imaging version k-t SENSitivity Encoding (k-t SENSE). Beyond the current scope it is expected that the information obtained from rapid MRI (cine-2D or 4D) can be merged with that from 3DCT or 4DCT to create a fused pretreatment 4D image that combines the soft-tissue contrast and temporally dense information from MRI with the spatial accuracy and electron density information from CT. Admittedly this is a nontrivial problem because one has to account for MRI artifacts correct for geometric distortions of the anatomy due to the relatively narrow bore of the magnet and develop robust multimodality image registration tools. Furthermore since this was a feasibility study the patients were not asked to lie in the treatment position for the MRI scan. However for future studies which aim to fuse the MRI with CT patients will be required to do so. However if these challenges are addressed fused 4D images would provide a more realistic picture of the behavior of thoracic anatomy over multiple respiratory cycles. Such guidance would enable the development of novel 4D treatment planning paradigms that explicitly account for effects such as baseline shifts and changes in abdominal versus thoracic breathing. Finally several investigators are working on integrated MRI+linac designs [21“23]. Online prospective 4D MRI would enable such systems to perform real-time monitoring and potentially real-time beam adaptation. 4. Conclusion We have investigated the feasibility of rapid MRI as a modality for image-based guidance in lung radiotherapy. While the acquisition speeds of cine-2D imaging are adequate for capturing most respiratory motion significant further improvements are required to achieve comparable speeds for truly 4D MRI acquisition. Nevertheless these early results indicate that rapid MRI offers a highly attractive noninvasive imaging tool for respiratory motion management. The ability to perform dose-free long-term monitoring over multiple respiratory cycles yields valuable information that is not currently available with 4DCT. We expect that such image-guidance will lay the groundwork for significantly better respiratory motion management in lung radiotherapy. Acknowledgments This work was partially supported by the American Association of Physicists in Medicine (AAPM) Research Seed Funding Grant 2008. Conflict of Interests The authors declare that there is no conflict of interests regarding the publishing of this paper. 1 Keall PJ Mageras GS Balter JM The management of respiratory motion in radiation oncology report of AAPM Task Group 76 Medical Physics 2006 33 10 3874 3900 2-s2.0-33749422038 17089851 2 Mageras GS Pevsner A Yorke ED Measurement of lung tumor motion using respiration-correlated CT International Journal of Radiation Oncology Biology Physics 2004 60 3 933 941 2-s2.0-4744343520 3 Keall P 4-Dimensional Computed Tomography Imaging and Treatment Planning Seminars in Radiation Oncology 2004 14 1 81 90 2-s2.0-0842306300 14752736 4 Wink NM Panknin C Solberg TD Phase versus amplitude sorting of 4D-CT data Journal of Applied Clinical Medical Physics 2006 7 1 77 85 2-s2.0-33645395992 16518319 5 Cai J Read PW Sheng K The effect of respiratory motion variability and tumor size on the accuracy of average intensity projection from four-dimensional computed tomography: an investigation based on dynamic MRI Medical Physics 2008 35 11 4974 4981 2-s2.0-54949144704 19070231 6 Yamamoto T Langner U Loo BW Jr. Shen J Keall PJ Retrospective analysis of artifacts in four-dimensional CT images of 50 abdominal and thoracic radiotherapy patients International Journal of Radiation Oncology Biology Physics 2008 72 4 1250 1258 2-s2.0-54049105620 7 Persson GF Nygaard DE Brink C Deviations in delineated GTV caused by artefacts in 4DCT Radiotherapy and Oncology 2010 96 1 61 66 2-s2.0-77953962041 20570002 8 Mori S Ko S Ishii T Nishizawa K Effective doses in four-dimensional computed tomography for lung radiotherapy planning Medical Dosimetry 2009 34 1 87 90 2-s2.0-58749115793 19181261 9 Plathow C Hof H Kuhn S Therapy monitoring using dynamic MRI: analysis of lung motion and intrathoracic tumor mobility before and after radiotherapy"

EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art

"differentiation of human stromal mesenchymal stem cells hMSCs is a critical procedure for thedevelopment of osteoblast SNHG14 is a newly discovered lncRNA that has been barely studied Our preliminaryexperiments showed that SNHG14 may be dysregulated in the differentiation of hMSCs In this study we focusedon elucidating the relationships among SNGH14 miR2861 and osteoblastic differentiation of hMSCsMethod To investigate the roles of SNHG14 and miR2861 in hMSCs differentiation qRTPCR luciferase activity celltransfections the detections of ALP activity and Alizarin Red staining were performedResult We found that the expression of SNHG14 was enhanced while the expression of miR2861 was suppressedin serum and hMSCs from patients with osteoporosis SNHG14 could target miR2861 and shSNHG14 suppressedosteoblast differentiation of hMSC MiR2861 suppressed osteoblast differentiation of hMSC In addition the effectsof SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861Conclusion In our experimental data showed that the induction effects of SNHG14 on osteoblastdifferentiation of hMSC were attenuated by miR2861 SNHG14 could induce osteogenic differentiation of hMSCin vitro by targeting miR2861Keywords SNHG14 Osteogenic differentiation Human stromal mesenchymal stem cells miR2861BackgroundMesenchymal stem cells have the capabilities of selfrenewal and multilineage differentiation which are critical factors in the regeneration or repairment of bone tissues [ ] Human bone marrow mesenchymal stem cellhMSCs could fully differentiate to many cell types including osteoblasts chondrocytes and adipocytes [ ]The differentiation of hMSCs is thus critical for the development of osteoblast Studies have modulated the cell signaling pathways to control the differentiation of hMSCs to Correspondence vs4190163com Mingchang Du and Bo Wu contributed equally to this workThe Orthopedic Hospital of Shenyang No Dong bei da ma lu road Dadong district of Shenyang Shenyang City Liaoning Province PRChinaosteoblasts [ ] However the underlying mechanismsremain to be elusiveNoncoding RNAs have become the hotspot in severalresearch fields including long noncoding RNAs lncRNAs nt [] and microRNAs miRNAs nt [] Various lncRNAs have been reported to be involved in theosteoblastic differentiation of hMSCs For instance downregulation of lncRNAANCR promoted osteoblast differentiation by targeting EZH2 and regulating the expression ofRunx2 [] LncRNA H19 was reported to mediate BMP9induced osteogenic differentiation of MSCs through theNotch signaling [] LncRNA SNHG14 is a newly discovered lncRNA that has been barely demonstrated regardingits biological roles in human diseases It was reported thatSNHG14 promoted microglia activation by regulating miR The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cDu BMC Musculoskeletal Disorders Page of 1455pPLA2G4A in cerebral infarction [] Very limitedinformation has been revealed for its functions in hMSCsMiRNAs are another group of noncoding RNAs thathave been widely reported in human diseases ManymiRNAs exert essential roles in the differentiation ofhMSCs to osteoblast For example microRNA138 wasrevealed to regulate the osteogenic differentiation of human stromal mesenchymal stem cells in vivo [] Another study also reported thatthe microRNA320RUNX2 axis regulates adipocytic differentiation of human mesenchymal skeletal stem cells [] MoreovermiR2861 has been demonstrated to participate in theregulatory feedback loop during differentiation of mouseosteoblast []From our preliminary experiment we noticed thatSNHG14 may be dysregulated in hMSCs differentiationand miR2861 may share the common binding sequenceswith lncRNA SNHG14 In this study we aimed to clarifythe role of lncRNA SNHG14 in the formation of osteoblast from hMSCs focusing on elucidating the relationshipsand osteoblasticdifferentiation of hMSCsamong SNGH14 miR2861MethodsHuman samplesIn this study patients with hip fracture were recruited atThe Orthopedic Hospital of Shenyang Patient sampleswere divided into two groups patients in each groupincluding the treatment group osteoporosis patientswith a fracture and the control group nonosteoporosispatients with a fracture Serum and bone tissues werecollected during endoprosthesis and gamma nail wasimplanted into the proximal femur All patients enrolledin this study signed the informed consent This studywas approved by the Research Ethics Committee of TheOrthopedic Hospital of ShenyanghMSC preparationshMSCs were obtained from the bone marrow from femurs of patients during total hip or knee arthroplastydue to osteoarthritis or hip fracture The Ethics ReviewBoard of Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study All hMSCswere obtained from postmenopausal women with anaverage age of years old age range “ years oldDensitometric examinations were performed using aLunar iDXA apparatus GE Lunar Madison WI USADiagnosis of oste ia or osteoporosis were made usingthe WHO Tscore criteria ˆ’ Tscore ˆ’ or Tscore ‰ ˆ’ respectively All the subjects in the osteoporosis group had vertebral fracturesCell separationThe RosetteSep Isolation kit STEMCELL Canada wasused to isolate hMSCs Cells were cultured at °C in awet environment with CO2 The culture mediumwas refreshed every week When cells reached confluence they were trypsinized and used immediatelyCell cultureWe cultured hMSCs in αminimum essential mediumαMEM containing fetal bovine serum FBS Invitrogen antibiotics and glutamax IGIBCO USAOsteogenesis was induced by fresh osteoblast inductionmedium OIM with ˆ’ M dexamethasone SigmaAldrich D4902 mM lascorbic acid SigmaAldrichA8960 mM glycerophosphateSigmaAldrichG9422 and mM 125vitaminD3 Alkaline phosphatase ALP was used to assess osteoblast phenotypeAlizarin Red staining was used to test matrixmineralization The medium was changed every dthroughout the experiments and cells were harvested atindicated time pointsqRTPCRTotal RNAs were extracted from serum bone tissues orhMSCs by Trizol Invitrogen USA The Reverse Transcription Kit Applied Bio USA was used to synthesizecDNAs The qRTPCR reactions were prepared usingSYBR Select Master Mix Applied Bio USA and PCRwas carried out on an ABI 7900fast thermocycler Applied Bio USA The relative expression was calculatedby 2ΔΔCT method The sequences of the primers arelisted belowSNHG14F ²GGGTGTTTACGTAGACCAGAACC3²SNHG14R ²CTTCCAAAAGCCTTCTGCCTTAG3²GAPDHF ²GAAGGTGAAGGTCGGAGTC3²GAPDHR ²GAAGATGGTGATGGGA TTTC3²OCF F ²GGCGCTACCTGTATCAATGG3²OCR ²GTGGTCAGCCAACTCGTCA3²Runx2F ²CGAATAACAGCACGCTATTAA3²Runx2R ²GTCGCCAAACAGATTCATCCA3²OSXF ²GCCAGAAGCTGTGAAACCTC3²OSXR ²GCTGCAAGCTCTCCATAACC3²ALPF ²TAGTGAAGAGACCCAGGCGCT3²ALPR ²ATAGGCCTCCTGAAAGCCGA3²miR2861F ²AACGAGACGACGACAGAC3²miR2861R ²GGGGCCUGGCGGUGGGCGG3²U6 ²GCCCCCGCCTCCGCCGCCGCC3² and ²ATATGGAACGCTTCACGAATT3²Cell transfectionsVectors with shSNHG14 miR2861 mimic and miR inhibitor all from Genepharma were transfectedto hMSCs via Lipofectamine Sigma USA At dposttransfection qRTPCR was conducted to detect 0cDu BMC Musculoskeletal Disorders Page of gene expressions The miR2861 mimic sequence was²GGGGCCUGGCGGCGGGCGG3² Mimic controlsequence was ²UUCUCCGAACGUGUCACGUTT3²The antagomir sequence was ²CCGCCCGCCGCCAGGCCCC3² The antagomir control sequence was ²CAGUACUUUUGUGUAGUACAA3²ALP activityhMSCs were collected and washed The cells were lysedby Triton X100 for min and centrifuged at g for min The supernatant was used for ALP analysis by ALP Assay Kit Abcam USAAlizarin red stainingThe osteoblasts were cultured by OIM for weeks andthen fixed by ethanol Next the cells were incubated by Alizarin Red solution for an hour at CThe results were recorded for analysisLuciferase assayPrimers were designed for the potential miR2861 binding sequence of AKT2 ²UTR SNHG14 ²UTR andthen cloned into the Sac IXba I sites of pmirGLODualLuciferase reporter vector The reconstructed plasmidswere confirmed by sequencing and named pmirGLOSNHG14WT and pmirGLOAKT2wt1 We also commercially synthesized mutant reporter constructs by mutating three nucleotides of each potential miR2861binding site and designated as pmirGLOSNHG14MUT pmirGLOAKT2mut1 Cells of confluencewere seeded in triplicate in 96well plates The wildtypeWT or mutant reporter constructs Mut were cotransfected into SiHa cells in the 96well plates with nmolL miR2861 or nmolL miRNC by using lipofectamine Invitrogen CA USA respectively Reporterposttransfection using the DualLuciferase Reporter AssayKit Promega following the manufacturer™s instructionsFirefly luciferase activity values were normalized fortransfection efficiency using the corresponding Renillaluciferase activity Three independent experiments wereperformedassays weregeneperformed hWestern blot analysisCell protein lysates were separated in or SDSPAGE gel h posttransfection followed by transferring to polyvinylidene difluoride membrane PVDFWestern blot analysis was performed with monoclonalantip53 Santa Cruz antiAKT2 Abcam primary antibodies AntiGAPDH antibody Santa Cruz was used asan internal control The membrane was washed and incubated with horseradish peroxidase HRPconjugatedsecondary antibody Cell Signaling Technology USAComplexes were visualized with SuperSignal West PicoChemiluminescent Substrate Pierce and the expressionlevels of these proteins were evaluated by Quantity OnesoftwareStatistical analysisData were shown as mean ± stand deviation SD Comparisons were performed by ttest between groups oroneway ANOVA among multiple groups P wasconsidered statistical significant differencesResultsSNHG14 was upregulated but miR2861 was downregulatedin serum and hMSCs from patients with osteoporosisThe expression of SNHG14 and miR2861 in serum andhMSCs of osteoporosis patients were analyzed Compared to participants without osteoporosis n theexpression levels of SNHG14 in serum and hMSCs ofn were greatly elevatedosteoporosis patiensFig 1a and c In addition the expression of miR2861was dramatically downregulated in hMSCs of osteoporosis group Fig 1d In addition a negative relationshipbetween the expression of SNHG14 and miR2861 in theserum of the osteoporosis group was observed Fig 1bfurtherinvestigated theSNHG14 was targeted by miR2861Werelationship betweenSNHG14 and miR2861 As shown in Fig 2a the common binding site between SNHG14 and miR2861 wasobserved After successfully transfecting miR2861 intohMSCs Fig 2b the cotransfection of SNHG14 ²UTR with miR2861 led to the suppression of luciferaseactivities compared with that of SNHG14 MUT Figue2C Moreover the transfection of shSNHG14 elevatedthe expression levels of miR2861 Fig 2d The expression levels of SNHG14 were also reduced in cells transfected with miR2861 Fig 2e Thees data indicated thatSNHG14 was targeted by miR2861reduced in cellsshSNHG14 suppressed osteoblast differentiation of hMSCTo investigate the effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiationto osteoblasts after transfection with shSNHG14 orshNC As shown in Fig 3a the expression levels ofSNHG14 weretransfected withshSNHG14 The suppression of SNHG14 markedly lowered osteoblastic differentiation which was indicated bylower expression levels of the osteoblastspecific genesRUNX2 Osterix OSX ALP OC and decreased ALP activity Figs 3bd We observed matrix mineralizationin vitro by Alizarin red staining in shSNHG14“transfected hMSCs compared with cells transfected withshNC It was obvious that shSNHG14 could suppresshMSCs differentiation to osteoblasts weeks posttransfection 0cDu BMC Musculoskeletal Disorders Page of Fig SNHG14 was upregulated but miR2861 was downregulated in serum and hMSCs from patients with osteoporosis a Expressions ofSNHG14 in the serum of nonosteoporosis people and osteoporosis patients n b The negative relationship between the expression ofSNHG14 and miR2861 in the serum of osteoporosis patients n c Expression of SNHG14 in hMSCs of nonosteoporosis people andosteoporosis patients n d Expression of miR2861 in hMSCs of nonosteoporosis people and osteoporosis patients n p Fig SNHG14 was targeted by miR2861 a Common binding sequences between SNHG14 and miR2861 b Expression of miR2861 mRNA inhMSCs c Dualluciferase reporter assay d Expression of miR2861 mRNA in hMSCs e Expression of SNHG14 mRNA in hMSCs N p 0cDu BMC Musculoskeletal Disorders Page of Fig shSNHG14 suppressed osteoblast differentiation of hMSC a The expression of SNHG14 mRNA in hMSCs b ALP activities in shSNHG14 orshNC transfected hMSCs on day day and day c Osteoblast differentiation assessed through osteoblast marker genes of RUNX2 OSX ALPand OC normalized to actin on day day and day d ALP and Alizarin Red staining on day N p MiR2861 suppressed osteoblast differentiation of hMSCTo further evaluate the effects of miR2861 on hMSCosteoblast differentiation we induced hMSCs to differentiate to osteoblasts after transfection with miR2861mimic or miRNC Overexpression of miR2861 significantly suppressed osteoblastic differentiation which wasindicated by decreased ALP activity Fig 4a lower expression levels of RUNX2 OSX ALP and OC Fig 4b 0cDu BMC Musculoskeletal Disorders Page of Fig MiR2861 suppressed osteoblast differentiation of hMSC a ALP activities measured at day day and day of osteoblastdifferentiation b osteoblast differentiation assessed by the mRNA expression of RUNX2 OSX ALP and OC day day and day c ALP andAlizarin Red staining results on day N p and reduced in vitro matrix mineralization Fig 4c inmiR2861mimic transfected hMSCs in contrast to cellstransfected with miRNCThe effects of SNHG14 on osteoblast differentiation ofhMSC were attenuated by miR2861Whether miR2861 could attenuate the effects ofSNHG14on osteoblast differentiation of hMSCFigure 5a illustrated that shSNHG14 decreased ALP activity but the effects were attenuated by cotransfectionwith miR2861 inhibitor Figure 5b demonstrated thatdownregulation of miR2861 greatly lowered osteoblastic differentiation induced by shSNHG14sinceshSNHG14 decreased osteogenesisAKT2 was targeted by miR2861Finally the mechanisms by which miR2861 functionedto affect the differentiation of hMSCs were exploredOur bioinformatics analysis and luciferase assay resultsshowed that AKT2 could bind with miR2861 Fig 6aand b In addition overexpression of miR2861 decreased the expression levels of AKT2 and downregulation of SNHG14 reduced the expression of AKT2Fig 6c and dDiscussionsOsteoblastic differentiation from hMSCs many originates from many cell events that are affected by variousmolecular and cellular procedures during the development of bone and skeleton It is crucial to reveal important factors that mediate this phenomenon and to studythe underlying mechanisms Owing to the successfulfindings from the previous studies different lncRNAshave been shown to participate in the osteoblast differentiation by targeting corresponding cell signaling pathways One study revealed the expression profiling of 0cDu BMC Musculoskeletal Disorders Page of Fig The effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861 a ALP activities in cells transfected withcontrol shSNHG14 or shSNHG14 miR2861inhibitor at day b Expression of osteoblast marker genes of RUNX2 OSX ALP and OC at day N p lncRNAs in C3H10T12 mesenchymal stem cells undergoing early osteoblast differentiation [] LncRNA H19promoted osteoblast differentiation via the TGF1Smad3HDAC signaling pathway by deriving miR675[]Various lncRNAs and miRNAs are dysregulated during the hMSCs differentiation of osteoblast [ ] Inour study we found a similar phenomenon We firstlyanalyzed the expression of SNHG14 and miR2861 inserum and hMSCs of osteoporosis patients Comparedto nonosteoporosis participants the expression levels ofSNHG14 in serum and hMSCs of osteoporosis patientswere greatly elevated The expression of miR2861 wasdrastically downregulated in hMSCs of osteoporosisgroup A negative relationship was established betweenthe expression of SNHG14 and miR2861 in serum ofosteoporosis group Similar to previous studies we identified that lncRNA SNHG14 was upregulated but miR was downregulated in serum and hMSCs from patients with osteoporosisFig AKT2 was targeted by miR2861 a Shared binding sequences between AKT2 and miR2861 b Dualluciferase reporter assay c and dWestern blot assay of AKT2 protein expression levels N p 0cDu BMC Musculoskeletal Disorders Page of With the common shared binding sequences lncRNAscould target their specific miRNAs and exert the biological roles in the pathogenesis of many cellular procedures [] For examplelncRNA DGCR5 acts as atumor suppressor in papillary thyroid carcinoma via targeting miR2861 [] We first confirmed the commonbinding sequences between SNHG14 and miR2861 Cotransfection of SNHG14 ²UTR with miR2861 led tothe suppression of luciferase activities compared withthat of SNHG14 MUT Moreover shSNHG14 elevatedthe expression levels of miR2861 The relative expression levels of SNHG14 were also lowered in cells transfected with miR2861 As far as we know we are thefirst to reveal that SNHG14 is targeted by miR2861 during the hMSCs differentiation to osteoblastAccording to previous reports ALP is highly expressedin osteoblast which is an important indicator for maturedifferentiation of osteoblast [] Osteoblastspecific genesRUNX2 Osterix ALP and OC are also critical genes to indicate the existing of osteoblast [ ] To investigatethe effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiation to osteoblastsafter transfection with shSNHG14 or shNC The expression of SNHG14 was suppressed in cells transfected withshSNHG14 Suppression of SNHG14 markedly loweredosteoblastic differentiation which was indicated by lowerexpression levels of the osteoblastspecific genes RUNX2Osterix ALP and OC decreased ALP activity and in vitromatrix mineralization by Alizarin red staining inshSNHG14 transfected hMSCs compared with cells transfected with shNC Similar to previous reports [ ] wealso observed that silencing of SNHG14 could suppresshMSCs differentiation to osteoblastsA novel regulation role of Runx2miR3960miR2861was demonstrated in mouse osteoblast differentiation []MiR2861 was found to promote osteoblast differentiationby increasing the expression of Runx2 [] To investigatethe effects of miR2861 on hMSC osteoblast differentiationwe induced hMSCs to differentiate to osteoblasts aftertransfection with miR2861mimic or miRNC Overexpression of miR2861 greatly suppressed osteoblastic differentiation which was indicated by lower expression levelsof the osteoblastspecific genes RUNX2 OSX ALP andOC and decreased ALP activity and reduced in vitromatrix mineralization in miR2861mimic transfectedhMSCs compared to cells transfected with miRNC Different from the previous study [] we noticed that miR2861suppressed osteoblast differentiation of hMSC Moreoverwe observed that the effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861ConclusionsIn our data confirmed that the induction effects of SNHG14 on osteoblast differentiation of hMSCswere attenuated by miR2861 SNHG14 could induceosteogenic differentiation of hMSC in vitro by targetingmiR2861Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035069Additional file AbbreviationhMSCs Human bone marrow mesenchymal stem cellAcknowledgmentsNot applicableIndividual persons dataNot applicableAuthors™ contributionsMD BW SF YL XM XF contributed to data analysis drafting or revising the gave final approval of the version to be published and agree to beaccountable for all aspects of the workFundingThere is no funding sourceAvailability of data and materialsThe analyzed data sets generated during the study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe ethics review board of the Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study Written informed consent wasobtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived January Accepted July ReferencesAggarwal S Pittenger MF Human mesenchymal stem cells modulateallogeneic immune cell responses Blood “Sonoyama W Liu Y Fang D Yamaza T Seo BM Zhang C Liu H GronthosS Wang CY Shi S Mesenchymal stem cellmediated functional toothregeneration in swine PLoS One 200611e79Nuttelman CR Tripodi MC Anseth KS Dexamethasonefunctionalized gelsinduce osteogenic differentiation of encapsulated hMSCs J Biomed MaterRes Part A “Dawson E Mapili G Erickson K Taqvi S Roy K Biomaterials for stem celldifferentiation Adv Drug Deliv Rev “Nguyen MK Jeon O Krebs MD Schapira D Alsberg E Sustained localizedpresentation of RNA interfering molecules from in situ forming hydrogels toguide stem cell osteogenic differentiation Biomaterials “Eskildsen T TaipaleenmÃki H Stenvang J Abdallah BM Ditzel N Nossent AYBak M Kauppinen S Kassem M MicroRNA138 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"As a result the presence of ALK gene translocation was confirmed (Figure 2F). The patient underwent crizotinib treatment (250 mg/bid orally) for 26 days. After this period symptoms including neck constriction and cough were improved. Chest CT scan images demonstrated decrease in tumor size and metastases. According to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1) such tumor response to crizotinib was classified as partial response (PR) (Figure 1C). Follow-up chest CT scan performed 11 weeks after the beginning of the treatment revealed a dramatic reduction in tumor size and mediastinal lymph node with nearly no presence of metastases in both lungs (Figure 1D). IL-6 (3.34 vs 25.41 pg/mL) and CEA (1.84 vs 23.43 ng/mL) levels were also reduced 23 weeks after the beginning of the therapy which demonstrated continuous partial response. Discussion According to the National Comprehensive Cancer Network guidelines ALK testing is not routinely performed in patients with squamous cell lung cancer. Therefore this patient was not tested for ALK until crizotinib was approved for marketing by the CFDA (June 23 2013). Unfortunately neither chemotherapy nor EGFR-TKI treatment produced effective tumor response in this patient. ALK gene translocations have been previously detected in patients with lung adenocarcinoma and light or non smoking history [34]. To date two studies have previously reported cases of patients with mixed carcinoma and squamous cell component harboring ALK rearrangements [56] but these studies did not describe specific therapy or diagnostic procedures. Another report showed that a patient with squamous cell lung cancer harboring c-MET amplification had partially responded to crizotinib [7]. Herein we report the case of a non-smoking woman with squamous cell lung cancer and ALK gene rearrangement who experienced remarkable response to crizotinib treatment after failed chemotherapy. In concordance with other studies on patients with lung adenocarcinoma treated with crizotinib [89] this patient has remained in remission (PR). Most importantly such remarkable response was obtained after two courses of failed chemotherapy. Conclusion Despite the low reconstruction rate of ALK gene if applicable patients with squamous cell lung cancer should have the option of undergoing ALK testing and receiving crizotinib treatment. ALK testing and subsequent targeted therapy could be an effective option for patients with non-small cell lung cancer who present progression following chemotherapy radiotherapy or any other treatment. Consent The patient provided written consent for publication of this case report. Abbreviations EML4: Echinodermmicro tubule associated protein like 4; ALK: Anaplastic lymphoma kinase; NSCLC: Non-small-cell lung cancer; CT: Chest computed tomography; TKIs: Tyrosine kinase inhibitors; EGFR: Epidermal growth factor receptor; IL-6: Interleukin-6; CEA: Carcinoembryonic antigen; CFDA: China Food and Drug Administration; FISH: Fluorescent in situ hybridization; PR: Partial response; IHC: Immunohistochemistry; H & E: Hematoxylin and eosin; ARMS: Amplification refractory mutation system; RECIST: Response Evaluation Criteria in Solid Tumors. Competing interests The authors declare that they have no competing interests. Authors™ contributions QSW carried out the molecular identifications of EGFR and K-ras and drafted the manuscript. YH provided parts of the clinical treatment data and revised the manuscript. XY participated in the H & E IHC and FISH analyses. YBW provided parts of the clinical treatment data. HLX participated in the design and coordination of this study and helped to revise the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/14/83/prepub Acknowledgements We would like to thank Li Lin for the assistance on IHC analysis. We would also like to thank Edanz Editing for language revision of the manuscript. Djalalov S Beca J Hoch JS Krahn M Tsao MS Cutz JC Leighl NB Cost effectiveness of EML4-ALK fusion testing and first-line crizotinib treatment for patients with advanced ALK-positive non-small-cell lung cancer J Clin Oncol 2014 32 10 1012 1019 10.1200/JCO.2013.53.1186 24567430 Rodig SJ Mino-Kenudson M Dacic S Yeap BY Shaw A Barletta JA Stubbs H Law K Lindeman N Mark E Janne PA Lynch T Johnson BE Iafrate AJ Chirieac LR Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population Clin Cancer Res 2009 15 5216 5223 10.1158/1078-0432.CCR-09-0802 19671850 Shaw AT Yeap BY Solomon BJ Riely GJ Gainor J Engelman JA Shapiro GI Costa DB Ou SH Butaney M Salgia R Maki RG Varella-Garcia M Doebele RC Bang YJ Kulig K Selaru P Tang Y Wilner KD Kwak EL Clark JW Iafrate AJ Camidge DR Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis Lancet Oncol 2011 12 11 1004 1012 10.1016/S1470-2045(11)70232-7 21933749 Inamura K Takeuchi K Togashi Y Nomura K Ninomiya H Okui M Satoh Y Okumura S Nakagawa K Soda M Choi YL Niki T Mano H Ishikawa Y EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers J Thorac Oncol 2008 3 1 13 17 10.1097/JTO.0b013e31815e8b60 18166835 Wong DW Leung EL So KK Tam IY Sihoe AD Cheng LC Ho KK Au JS Chung LP Pik Wong M The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS Cancer 2009 115 1723 1733 10.1002/cncr.24181 19170230 Shaw AT Yeap BY Mino-Kenudson M Digumarthy SR Costa DB Heist RS Solomon B Stubbs H Admane S McDermott U Settleman J Kobayashi S Mark EJ Rodig SJ Chirieac LR Kwak EL Lynch TJ Iafrate AJ Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK J Clin Oncol Off J Am Soc Clin Oncol 2009 27 4247 4253 10.1200/JCO.2009.22.6993 Schwab R Petaka I Kollar M Pinter F Varkondi E Kohank A Barti-Juhasz H Sch¶nleber J Brauswetter D Kopper L Urban L Major partial response to crizotinib a dual MET/ALK inhibitor in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement Lung Cancer 2014 83 1 109 111 10.1016/j.lungcan.2013.10.006 24192513 Toyokawa G Takenoyama M Watanabe S Toyozawa R Inamasu E Kojo M Shiraishi Y Morodomi Y Takenaka T Hirai F Yamaguchi M Taguchi K Seto T Ichinose Y Dramatic response to crizotinib in an ALK-positive adenocarcinoma patient with disseminated intravascular coagulation J Thorac Oncol 2013 8 11 e96 e98 10.1097/JTO.0b013e3182a008ed 24128725 Kim SJ Kim DW Kim TM Lee SH Heo DS Bang YJ Remarkable tumor response to crizotinib in a 14-year-old girl with ALK-positive non“small-cell lung cancer J Clin Oncol 2012 30 16 e147 e150 10.1200/JCO.2011.39.9766 22508824 15030100R 648 Ann Thorac Surg Ann. Thorac. Surg. The Annals of thoracic surgery 0003-4975 1552-6259 25106680 4185369 10.1016/j.athoracsur.2014.05.026 NIHMS619453 Intraoperative Near-Infrared Imaging Can Identify Pulmonary Nodules Okusanya Olugbenga T. MD 1 Holt David 2 Heitjan Daniel 3 Deshpande Charuhas 4 Venegas Ollin 1 Jiang Jack 1 Judy Ryan 1 DeJesus Elizabeth 1 Madajewski Brian 1 Oh Kenny Albelda Steven M. 5 Nie Shuming 6 Singhal Sunil MD 1 5 * 1Division of Thoracic Surgery Department of Surgery University of Pennsylvania School of Medicine Philadelphia Pennsylvania 2Department of Clinical Studies University of Pennsylvania School of Veterinary Medicine 3Department of Biostatistics & Epidemiology University of Pennsylvania 4Department of Pathology University of Pennsylvania School of Medicine 5Department of Medicine University of Pennsylvania School of Medicine 6Departments of Biomedical Engineering and Chemistry Emory University Atlanta Geia *Corresponding Author: Sunil Singhal Division of Thoracic Surgery University of Pennsylvania School of Medicine 6 White Building 3400 Spruce Street Philadelphia PA 19104 sunil.singhaluphs.upenn.edu 9 8 2014 5 8 2014 10 2014 01 10 2015 98 4 1223 1230 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved. 2014 Background Over 80000 people undergo pulmonary resection for a lung nodule in the United States each year. Small nodules are frequently missed or difficult to find despite preoperative imaging. We hypothesized that near-infrared (NIR) imaging technology could be used to identify and locate lung nodules during surgery. Methods We enrolled 18 patients who were diagnosed with a pulmonary nodule that required resection. All patients had a fine-cut 1mm computed tomography scan preoperatively. The patients were given systemic 5 mg/kg indocyanine green (ICG) and then underwent an open thoracotomy 24 hours later. NIR imaging was used to identify the primary nodule and search for additional nodules that were not found by visual inspection or manual palpation of the ipsilateral lung. Results Manual palpation and visual inspection identified all 18 primary pulmonary nodules and no additional lesions. Intraoperative NIR imaging detected 16 out of the 18 primary nodules. NIR imaging also identified 5 additional subcentimeter nodules: 3 metastatic adenocarcinomas and 2 metastatic sarcomas. This technology could identify nodules as small as 0.2 cm and as deep as 1.3 cm from the pleural surface. This approach discovered 3 nodules that were in different lobes than the primary tumor. Nodule fluorescence was independent of size metabolic activity histology tumor grade and vascularity. Conclusions This is the first-in-human demonstration of identifying pulmonary nodules during Thoracic surgery with NIR imaging without a priori knowledge of their location or existence. NIR imaging can detect pulmonary nodules during lung resections that are poorly visualized on computed tomography and difficult to discriminate on finger palpation. Mol Cancer Mol. Cancer Molecular Cancer 1476-4598 BioMed Central 24655544 3998010 1476-4598-13-68 10.1186/1476-4598-13-68 Research Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition Sun Ming 1 sunmingnjmu.edu.cn Liu Xiang-Hua 1 liuxianghuanjmu.edu.cn Wang Ke-Ming 2 422825636qq.com Nie Feng-qi 3 957714486qq.com Kong Rong 1 31815857qq.com Yang Jin-song 4 yangjinsongmedmail.com.cn Xia Rui 1 273459189qq.com Xu Tong-Peng 3 1034045558qq.com Jin Fei-Yan 1 759729211qq.com Liu Zhi-Jun 1 sm13776403108126.com Chen Jin-fei 4 1423594097qq.com Zhang Er-Bao 1 273459189qq.com De Wei 1 deweinjmu.edu.cn Wang Zhao-Xia 2 zhaoxiawang88hotmail.com 1Department of Biochemistry and Molecular Biology Nanjing Medical University Nanjing 210029 People™s Republic of China 2Department of Oncology Second Affiliated Hospital Nanjing Medical University Nanjing Jiangsu 210011 People™s Republic of China 3Department of Oncology First Affiliated Hospital Nanjing Medical University Nanjing People™s Republic of China 4Department of Oncology Nanjing First Hospital Nanjing Medical University Nanjing P. R. China 2014 21 3 2014 13 68 68 16 9 2013 13 3 2014 Copyright 2014 sun et al.; licensee BioMed Central Ltd. 2014 sun et al.; licensee BioMed Central Ltd. This is an Open Access distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this unless otherwise stated. Background Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cellular processes such as differentiation proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. The clinical significance of BANCR and its™ molecular mechanisms controlling cancer cell migration and metastasis are unclear. Methods Expression of BANCR was analyzed in 113 non-small cell lung cancer (NSCLC) tissues and seven NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of BANCR in NSCLC cells. The effects of BANCR on cell viability were evaluated by MTT and colony formation assays. Apoptosis was evaluated by Hoechst staining and flow cytometry. Nude mice were used to examine the effects of BANCR on tumor cell metastasis in vivo. Protein levels of BANCR targets were determined by western blotting and fluorescent immunohistochemistry. Results BANCR expression was significantly decreased in 113 NSCLC tumor tissues compared with normal tissues. Additionally reduced BANCR expression was associated with larger tumor size advanced pathological stage metastasis distance and shorter overall survival of NSCLC patients. Reduced BANCR expression was found to be an independent prognostic factor for NSCLC. Histone deacetylation was involved in the downregulation of BANCR in NSCLC cells. Ectopic expression of BANCR impaired cell viability and invasion leading to the inhibition of metastasis in vitro and in vivo. However knockdown of BANCR expression promoted cell migration and invasion in vitro. Overexpression of BANCR was found to play a key role in epithelial-mesenchymal transition (EMT) through the regulation of E-cadherin N-cadherin and Vimentin expression. Conclusion We determined that BANCR actively functions as a regulator of EMT during NSCLC metastasis suggesting that BANCR could be a biomarker for poor prognosis of NSCLC. Background Non-small cell lung cancers (NSCLCs) including adenocarcinomas and squamous cell carcinomas are the predominant forms of lung cancer and account for the majority of cancer deaths worldwide [1]. Despite recent advances in clinical and experimental oncology the prognosis of lung cancer remains poor with a 5-year overall survival rate of around 11% [2]. A continuing problem of NSCLC tumorigenesis is the metastasis of cancer cells which is the main cause of death in patients. Thus a detailed understanding of the mechanisms and molecular pathways activated in metastatic cells is crucial in identifying new treatment options for anticancer therapy that target metastasis. The invasion and metastasis of cancer cells are landmark events that involve many changes in cellular behavior and lead to different steps of the metastatic cascade [34]. One of the most crucial steps in the metastatic cascade is the acquisition of invasive capabilities including turnover of cell-cell junctions degradation of the cell matrix and activation of pathways that control cytoskeletal dynamics in cancer cells. This process is accompanied by multiple changes in gene expression such as the loss of epithelial markers and a gain in mesenchymal markers [56]. Over the past decade cell and tumor biologists have identified the key role of epithelial-mesenchymal transition (EMT) in cancer cell metastasis a biological process where epithelial cells lose their polarity and undergo transition into a mesenchymal phenotype [7]. EMT enhances tumor cell invasion in response to environmental triggers and augments invasive functions by promoting Rac-dependent mesenchymal migration and also contributes to cell growth and survival [89]. Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of non-epithelial cadherins such as N-cadherin. The loss of E-cadherin expression is a fundamental event in EMT and a crucial step in the progression of papillomas to invasive carcinomas [10]. To date substantial effort has been devoted to understanding how EMT is regulated during cancer progression. It has been verified that EMT can be initiated by external signals such as hepatocyte growth factor (HGF) epidermal growth factor (EGF) transforming growth factor (TGF)-b and fibroblast growth factor (FGF) [11]. In addition to these signaling pathways triggered by membrane receptors recent studies have highlighted the importance of noncoding RNAs in the regulation of the epithelial phenotype by controlling EMT inducers. The miR-200 family has been found to control EMT by downregulating the expression of Zeb factors [12]. Furthermore the long noncoding RNA (lncRNA) MALAT-1 promoted EMT by regulating ZEB1 ZEB2 and Slug expression and activating Wnt signaling [13]. The lncRNAs are important new members of the ncRNA family that are greater than 200 nt and are unable to be translated into proteins. These lncRNAs are often expressed in a spatial- and temporal-specific pattern. Although very few lncRNAs have been characterized in detail they have been found to participate in a large range of biological processes including modulation of apoptosis and invasion reprogramming stem cell pluripotency and parental imprinting. These findings indicate that lncRNAs play a major role in the regulation of the eukaryotic genome [14-16]. Researchers have linked the dysregulation of lncRNAs with diverse human diseases in particular cancers [17-19]. Therefore identification of cancer-associated lncRNAs and investigation of their molecular and biological functions in controlling EMT are important in understanding the molecular biology of NSCLC metastasis and progression. BRAF-activated non-coding RNA (BANCR) an 693-bp lncRNA on chromosome 9 was firstly found by Ross J. Flockhart et.al via RNA-seq screen for transcripts affected by the expression of the oncogene BRAFV600E. BANCR is overexpressed in melanoma cells and crucial for melanoma cell migration [20]. In this study we investigated the effects of BANCR expression on NSCLC cell phenotypes in vitro and in vivo. Moreover we also showed that alteration of BANCR expression can influence E-cadherin N-cadherin and Vimentin protein levels which indicated that BANCR affected NSCLC cells invasion and metastasis partly via epithelial-mesenchymal transition. This study advances our understanding of the role of lncRNAs such as BANCR as a regulator of pathogenesis of NSCLC and facilitate the development of lncRNA-directed diagnostics and therapeutics. Results BANCR expression was downregulated and correlated with poor prognosis of NSCLC BANCR expression levels were investigated in 113 paired NSCLC samples and adjacent histologically normal tissues using quantitative polymerase chain reaction (qPCR) assays. BANCR expression was significantly downregulated (P?<?0.01) in 79% (89/113) of cancerous tissues compared with normal tissues (Figure 1A). BANCR expression levels in NSCLC were significantly correlated with tumor size (p?=?0.001) advanced pathological stage (p?<?0.001) and lymph node metastasis (p?=?0.001). However BANCR expression was not associated with other parameters such as gender (p?=?0.232) and age (p?=?0.616) in NSCLC (Table 1). The clinical data for all patients were summarized in Additional file 1: Table S1. Figure 1 Relative BANCR expression in NSCLC tissues and its clinical significance. (A) Relative expression of BANCR in NSCLC tissues (n?=?113) compared with corresponding non-tumor tissues (n?=?113). BANCR expression was examined by qPCR and normalized to GAPDH expression. Results were presented as the fold-change in tumor tissues relative to normal tissues. (B) BANCR expression was classified into two groups. (C D) Kaplan“Meier disease-free survival and overall survival curves according to BANCR expression levels. *P?<?0.05 **P?<?0.01. Table 1 Correlation between BANCR expression and clinicopathological characteristics of NSCLC patients (n?=?113) Characteristics BANCR P High no. cases (%) Low no. cases (%) Chi-squared test P-value Age(years) 0.616 ?65 29(54.7) 30(50.0) >65 24(45.3) 30(50.0) Gender 0.232 Male 35(66.0) 33(55.0) Female 18(34.0) 27(45.0) Histological subtype 0.466 Squamous cell carcinoma 30(56.6) 38(63.3) Adenocarcinoma 23(43.4) 22(36.7) TNM Stage "

as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is confirmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors “ mnx1 was firstly foundto be expressed in lymphoid and pancreatic tissues and definedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia “ furthermore recent studiesconfirmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer “the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these findingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression profiling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at —¦c in a humidified incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the affiliated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were confirmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university affiliated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraffinembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was confirmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the finalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturer™s protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcramplified and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the œcell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after “ days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at —¦c and fixed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with × clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of × hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using fluorescencemicroscopy and proliferation cell ratios were counted fromfive random fields in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were fixedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artificial scratch on a confluent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were filled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were fixed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from five random fields onevery filter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension × cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the œcell index valuecell cycle analysiscells were digested with trypsinedta and fixed with ethanol for h at —¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at —¦c a facscalibur flow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0“g1 s and g2“m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientific and scraped into a centrifuge tube the cells were centrifugedfor min at g at —¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at × g —¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at—¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at —¦c the chip elution buï¬ercontaining proteinase k was used for dna purification thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr amplification with primersspecific to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region ˆ’ bp wasamplified and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice “ weeks old were purchased from nanjingmedical university school of medicine™s accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group × exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the specification of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length × width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression profiling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients™ clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage i“ii vs iii“iv p figure 1f t stage t1 vst2“t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ‰¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two specific sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression efficiency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 confirmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3c“g of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in œtranscriptionand œmetabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been confirmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no significance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines c“f two specific sirna si1 and si2 of mnx1 were designed and the transfection efficiencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot g“i the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefficiencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no significancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespecific dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which “ bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by flow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no significanceaacaataaat and “ bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efficiency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments b“d the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efficiency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacrificed and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacrificed andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no significancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection efficiency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6b“f and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown efficiency wasconfirmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7c“e ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7h“kfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identified mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourfindings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentified to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebrafish and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer “for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also confirmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identified as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identified as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 “thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i

"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ‰¥ and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ‰¥ for the four most common cancers and ‰¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ‰¥ for the four most common cancers and ‰¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study –º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers –º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions –º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data –º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5“ the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprd™s linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 œmalignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures “figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ‰¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures “ post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ‰¥ data sources captured cancers close to the gold standard date median lag ‰¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep ht“htrqi inademelitnecreprqi inadem ht“htelitnecrep ht“ht inademrqielitnecrep ht“htcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ““ ot ot ot cl amoeym epitluml ot ci ameakuel““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot “ot “““““““ ot ot ot ot ot ot ot ““““““““““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““ˆ’““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““““““ inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep ht“ht““““““““““““““sarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot “ ot ot ot ot “““““c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprd™s recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncras™s recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu

"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ‰¥ and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ‰¥ for the four most common cancers and ‰¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ‰¥ for the four most common cancers and ‰¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study –º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers –º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions –º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data –º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5“ the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprd™s linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 œmalignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures “figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ‰¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures “ post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ‰¥ data sources captured cancers close to the gold standard date median lag ‰¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep ht“htrqi inademelitnecreprqi inadem ht“htelitnecrep ht“ht inademrqielitnecrep ht“htcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ““ ot ot ot cl amoeym epitluml ot ci ameakuel““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot “ot “““““““ ot ot ot ot ot ot ot ““““““““““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““ˆ’““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““““““ inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep ht“ht““““““““““““““sarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot “ ot ot ot ot “““““c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprd™s recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncras™s recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu

Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed women™s malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplan“Meier‘plotter database analysisKaplan“Meierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionP‘valueAge ‰ Tumor size ‰ Lymph node metastasis Present AbsentHistopathological grade I“II IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRT‘PCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ˆ’ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stably‘overexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine„¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures™ instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK‘ assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells × stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then × stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Student™s ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using Kaplan“Meierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by Kaplan“Meier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3c“f Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04c“q However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09c“f Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer c“q The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by Kaplan“Meierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010g“j and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ “] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro a“b The overexpression effect of GPX3overexpressed plasmid in breast cancer cells c“d Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells e“f Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network c“f The prognostic values of four miRNAs in breast cancer g“j The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pRˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ P‘valueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRP‘valuepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors™ contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics “ Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging “ Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol “ An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci “ Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res “ Kulis M Esteller M DNA methylation and cancer Adv Genet “ Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget “ Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res “ Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy “ Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol “ Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res “ Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie

"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[“] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a specific gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identified as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identified and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly defined erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgf𝛽p smad3 smad3testosterone𝛽catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebp𝛽mapkg3k3𝛽p2cebp𝛽cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis forced expression ofpparÎ is sufficient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincfinger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function efficientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparΓ“ï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeficient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase fibroblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the first klf family members which were identified to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic fibroblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebp𝛽cebp𝛿gata23ppará½»cebp𝛼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory protein“protein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to specificallyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown histone modification in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding affinity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [“] lysinespecific histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modification of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespecific histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a specific proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage specificity adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the ‚uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 “ mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebp𝛽 geneebf1 geneklf4egr2cebp𝛽cytosolcebp𝛿 genecebp𝛿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebp𝛼 geneznf638znf467cebp𝛼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgf𝛽1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods “ ““ and “ days and only autophagy inhibition at “days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is ‚uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes specific to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deficient mice the fifth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr

antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to find cooccurrence profiles of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions betweencommensal gut bacteria and a specific pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difficult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a œhigh priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut flora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection specifically b producta”a colonizer ofthe colon”reduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual benefits during pathogenpersistence these findings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene profiling analyses highlighted a firstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are specific murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene databœ“œ“œ“«º«ºœ“œ“œ“œ“œ“«º«ºœ“œ“œ“metabolomicsbœ“«º«º«º«ºœ“œ“œ“œ“œ“«º«º«º«ºœ“avg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols œ“ sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of five and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed firstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community profiles wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rarefied to reads alpha rarefaction analysis demonstratedsufficient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity profiles of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community profiling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9figshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faith™s phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identifies sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical confidence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faith™s phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the five most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the five most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classified as enterococcus”and identified as the most positively associatedwith the postvrefm colonization phase”was most abundant on days of challengeconfirming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classified as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identified an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identifies differential metabolome profiles duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of flight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in œmetabolic handoffs that define interspecies interactions analysis of the globalmetabolome profile of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteprofiles along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum profiles from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique profiles of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±–10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantification values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identified ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic profiles samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classifier identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identifications abx tx antibiotictreatment c peak quantification values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantification values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct profile shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentification of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classified to the same taxonomicrank for example our analyses identified multiple esvs classified as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further firstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen œbaseline microbiota profiles of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome profiles that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identified via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identified are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identified bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspecific microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent œsuppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens “ further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that define interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourfindings add support to the incorporation of microbiome profiling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are fixed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturer™s protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was amplified using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene profiling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identification ofsotus [] the output biom files were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rarefied to reads per sampletaxonomy was assigned using the sklearn classifier and greengenes otus from 515f806rregion of sequences classifier available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannon™s diversityindex faith™s phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute first distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal firstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsignificance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classifier analyses of input samples wereallocated to train the random forest classifier within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at “ °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of flight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identificationmetabolomic analyses

"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [“] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitin“proteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesforŽ corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury “ types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16ˆ’ monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [“] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1ˆ’ mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu

"Interferon COVID19SARSCOV2IranIn this study efficacy and safety of interferon Interferon beta1-b in the treatment of patients with severe COVID19 wereevaluatedAmong an label randomized clinical trial adult patients ‰¥ years old with severe COVID19 wererandomly assigned to the IFN group or the control group Patients in the IFN group received Interferon beta1-b mcg subcutaneously every other day for two consecutive weeks along with the national protocol medicationswhile in the control group patients received only the national protocol medications lopinavirritonavir oratazanavirritonavir plus hydroxychloroquine for “ days The primary outcome of the study was time toclinical improvement Secondary outcomes were inhospital complications and 28daymortalityBetween April and May patients were enrolled and finally patients in each group completed the study Time to clinical improvment in the IFN group was significantly shorter than the control group[“ vs “ days respectively p HR CI “] At day the percentageof discharged patients was and in the IFN and control groups respectively OR CI“ p ICU admission rate in the control group was significantly higher than the IFN group vs p The duration of hospitalization and ICU stay were not significantly differentbetween the groups Allcause 28day mortality was and in the IFN and control groups respectively p Interferon beta1-b was effective in shortening the time to clinical improvement without serious adverse events inpatients with severe COVID19 Furthermore admission in ICU and need for invasive mechanical ventilationdecreased following administration of Interferon beta1-b Although 28day mortality was lower in the IFN group furtherrandomized clinical trials with large sample size are needed for exact estimation of survival benefit of Interferon beta1-b IntroductionCoronavirus disease CoVID19 was reported from Wuhan forthe first time in late December Causing severe acute respiratorysyndrome coronavirus 2SARSCoV2 [] it rapidly spread throughoutthe world to the extent that the World Health anization WHOstated it as pandemic in March [] Until July more than million confirmed cases of CoVID19 were reported worldwideFurthermore more than deaths were recorded []Until now there is no definite antiviral treatment for CoVID19 andattempts continue for finding effective treatments worldwide Howeverfrom the beginning of the pandemic various treatments such as antiretrovirals antimalaria agents favipiravir remdesivir and corticosteroids immunoglobulin and cytokine blockers as adjunctive therapieswere suggested for the treatment of CoVID19 [] Except for the remdesivir which has had acceptable results the efficacy of other drugshas not been significant on the outcomes of the patients with CoVID19[“]Interferons IFNs have a key role in defense against viral infectionsas a component of innate immune system [] Invitro activity of IFN Ž Corresponding author at Department of Pharmacotherapy Tehran University of Medical Sciences Tehran IranEmail addresses khalilihtumsacir Khalilihsinatumsacir H Khalili101016jintimp2020106903Received July Received in revised form July Accepted August Available online August Elsevier BV All rights reserved 0cInternational Immunopharmacology prophylaxis deep vein thrombosis treatment of electrolyte disordersand antibiotic therapy were considered according to the hospital protocols The duration of the study was two weeks A 4week followupperiod was considered for all patientsPatients™ demographic data baseline diseases symptoms at the timeof disease presentation vital signs and laboratory data at the time ofhospital admission were recorded Patients were daily monitored interms of changes in the vital signs hemodynamic parameters oxygenation status laboratory data and treatment strategies Clinical statusof the patients was assessed by the sixcategory ordinal scale at days and of the randomization [] Need for supplemental oxygentherapy and also invasive or noninvasive respiratory supports wereevaluated regularly OutcomesTime to clinical improvement was considered as primary outcome ofstudy Clinical improvement was defined as improvement of at leasttwo points from the baseline status on the sixcategory ordinal scale[] This scale contains the subsequent categories death hospital admission requiring invasive mechanical ventilation hospitaladmission requiring noninvasive positive pressure ventilation hospital admission requiring oxygen hospital admission not requiring oxygen discharge Secondary outcomes were clinical statusof patients at day and ICU admission and intubation rateslength of hospitalization and ICU stay and 28day mortalitySide effects related to IFN therapy and other adverse events duringthe study period were monitored and recorded as the safety outcomesCategorization of adverse events was done according to the commonterminology criteria for adverse events CTCAE National Institutes ofHealth and National Cancer Institute Also serious complications during the hospitalization course such asacute respiratory distress syndrome ARDS nosocomial infectionsseptic shock acute kidney injury AKI and acute hepatic injury AHIwere considered Statistical analysis and randomizationContinuous variables are demonstrated as median interquartilerange IQR and categorical variables as frequencies and percentagesContinuous variables were compared between the groups by MannWhitney U test The Fisher™s exact test was applied for comparison ofcategorical variablesThe Hazard Ratio HR and CI for clinical improvement wereestimated by Cox proportional hazards regression analysis The effect ofischemic heart disease lymphocyte count Aspartate aminotransferaseAST and Creactive protein CRP on the primary outcome was evaluated by the adjusted Cox regression models as potential confoundingfactors Time to clinical improvement was estimated by KaplanMeierplot and compared with a logrank test All statistical analysis was doneby SPSS software version Time to clinical improvement was estimated to be approximately days and sample size was calculated by following equationH Rahmani et alhas been shown against severe acute respiratory syndrome coronavirusSARSCoV and Middle East respiratory syndrome coronavirus MERSCoV [“] Although IFN was used less than IFN α for the treatment of SARSCOV and MERSCoV in human studies it was effective inthe treatment of MERSCoV in retrospective studies and case series[“] The efficacy of Interferon beta1-b is being assessed in the treatment ofMERS in a randomized clinical trial [] According to the presence ofthis evidence IFN was considered as a promising option for thetreatment of CoVID19In this label randomized clinical trial efficacy and safety ofInterferon beta1-b in the treatment of patients with severe CoVID19 were assessed Materials and methods Study designThis label randomized clinical trial was designed to evaluatethe efficacy and safety of Interferon beta1-b in the treatment of patients withCoVID19 Patients with severe CoVID19 who were hospitalized duringApril to May in Imam Khomeini Hospital Center one ofthe largest referral hospitals in Tehran Iran were includedThe protocol of the study was approved by Ethics Committee ofTehran UniversityReferencenumberIRTUMSVCRREC13981053 Furthermore the study was registeredas a clinical trial register ID IRCT20100228003449N27 The studyprotocol was described for participants and written informed consentswere obtained from all patients or their firstdegree family members Eligibility criteriaof MedicalSciencesSARSCoV2 in patients™ nasopharyngeal swabs was detected usingRealTime Polymerase Chain Reaction RTPCR Total RNA extractionwas done applying Viral Nucleic Acid Extraction kit Cat No YVN50YVN100 from RBC Bioscience Taipei Taiwan The Novel Coronavirus2019nCOV Nucleic Acid Diagnostic Kit PCRFluorescence Probingof Sansure Biotech S3102E Changsha China was used for RTPCRAdult patients ‰¥ years old with positive PCR and clinicalsymptomssigns of pneumonia including dyspnea cough and feverperipheral oxygen saturation SPO2 ‰ in ambient air or arterialinspired oxygen PaO2oxygen partial pressure to fractionalFiO2 or SPO2FiO2 and lung involvement in chestimaging were included These criteria indicated severe form of thedisease [] At baseline patients with serious allergic reactions to IFNhistory of suicide thoughts and attempts alanine amino transferaseALT × the upper limit of the normal range uncontrolled underlying diseases such as neuropsychiatric disorders thyroid disorderscardiovascular diseases and also pregnant and lactating women werenot includedRecruitment was considered during the first 48hour of the hospitaladmission During the study period patients who received less than doses of Interferon beta1-b were excluded If patients were discharged beforefulfilment of the treatment course the treatment was applied at home ProceduresEligible patients were recruited in the IFN group or the controlgroup according to the permuted block randomization Patients in theIFN group received Interferon beta1-b along with the national protocol medications while in the control group patients received only the nationalprotocol medications Interferon beta1-b Ziferon® Zist Daru Daneh Co Iranwas administrated as mcg subcutaneously every other day for twoconsecutive weeks The national protocol consisted lopinavirritonavir mg BD or atazanavirritonavir mg daily plushydroxychloroquine mg BD in first day and then mg BD for“ days Other supportive cares such as fluid therapy stress ulcernkzz1122211222nn121512005015Z19612Z1041 0cH Rahmani et alInternational Immunopharmacology Fig Consort flowchart of the studyAccording to the above equation at least patients in each groupwere expected to make a difference of days in time to clinical improvement with power of Patients were randomly recruited to the IFN group or the control group The method of randomizationwas the permuted block randomization patients per block A biostatistician who was not involved in patients™ care did this process Results PatientsA total of patients were screened Of them patients did nothave the eligibility criteria of study and patients were referred fromanother hospital Three and four patients withdrew the consent duringthe study in the IFN group and control groups respectively Four patients did not adhere to IFN injection after second or third dose Alsothree patients in the control group were enrolled in another trialFinally patients in each group completed the study Fig The median IQR age of patients was “ years and of them were male No significant difference in terms of the patients™demographic data was detected between the groups The most commoncomorbidities were hypertension diabetes mellitus and ischemic heartdisease Dyspnea fever and cough were the most frequent symptoms atthe time of hospital admission The median IQR time from onset of thesymptoms to hospital admission was “ and “ days in the IFNgroup and control groups respectively The time from onset of thesymptoms to randomization was not statistically significant betweenthe groups All of patients required respiratory support at the time ofrandomization Oxygenation through facemask was required for morethan percent of patients None of the patients in both groups wereintubated at baseline Table Vital signs and laboratory data of patients at the time of recruitment were comparable between the groupsTable During the hospitalization course oxygen saturation droppedin and of patients in the IFN and control groups respectively All of those patients were intubated At least one antibioticwas administrated for and of patients in the IFN groupand control groups respectively Methylprednisolone was administeredfor of patients in the IFN group and of patients in the4Power085 0cH Rahmani et alTable Baseline characteristics of patientsParameter Median IQR or n AgeSexMaleFemaleComorbid conditions nHypertensionDiabetes mellitusIschemic heart diseaseAsthmaCOPDMalignancyTransplantationSymptoms at admission nDyspneaFeverCoughChillsDuration of symptoms before admissionmedian IQR daysTime from symptom onset torandomization median IQR daysSix category scale at day of intervention3hospital admission requiring highflownasal cannula or noninvasivemechanical ventilationsupplemental oxygen hospital admission requiringInterferon groupn “ ““Control groupn “ ““control group The dose of methylprednisolone was mg daily for days Methylprednisolone was considered during the cytokine orhyperinflammation phase days “ of onset of the symptoms Approximately and of patients in the INF and control groupsneeded vasopressors during the hospitalization course respectivelyTable Primary outcomesThe time to clinical improvement in the IFN group was significantlyshorter than the control group [“ vs “ days respectivelyp ] Table Moreover the Cox proportional hazards regression analysis showed that time difference to clinical improvement wasstatistically significant between the groups HR CI“ Fig Then the model was adjusted for the confoundingInternational Immunopharmacology Interferon groupn Control groupn Table Respiratory support and medicationsParameter n Respiratory supportNasal cannulaFace maskNIPPVIMVAntibiotics mer em piperacillintazobactam ceftriaxone FQsvancomycin azithromycin andColistin n CorticosteroidsVitamin CVasopressorsDiphenhydramineCardiovascular drugsStatinsARBsBetablockersACEIsNIPPV noninvasive positive pressure ventilation IMV invasive mechanicalventilation FQs fluoroquinolones ARB Angiotensin Π Receptor Blocker ACEIangiotensin converting enzyme inhibitorfactors and similar results were seen HR CI “ Secondary outcomesAccording to the six category scale and of patientswere discharged in the IFN and the control groups at day respectivelyOR CI “ p Only one patient in thecontrol group died at day Also at this time and patients wereintubated in the IFN and control groups respectively At day thepercentage of discharged patients reached to and in theIFN and control groups respectively OR CI “p Furthermore the number of deaths increased to and patients the IFN and control groups respectively Finally at day ofinclusion the proportion of discharged patients were in the IFNgroup and in the control group OR CI “p At this time ICU admission rate in the control group wassignificantly higher than the IFN group vs p Moreover more patients in the control group needed invasive mechanical ventilation compared with the IFN group but the rate was notstatistically different p Although length of hospitalization wasTable Patients™ vital signs and laboratory data at the time of hospital admissionParameter Median IQRTemperature °CHeart rate beats minuteRespiratory rate breathsminSystolic blood pressure mm HgSPO2 Laboratory dataWhite Blood Cell cells μlAcute Lymphocyte count cellsμlHemoglobin gdlPlatelet count cells × 103μlBlood Urea Nitrogen mgdlCreatinine mgdlAspartate aminotransferase ulAlanine aminotransferase ulAlkaline phosphatase ulTotal bilirubinmgdlCreactive protein mgdlErythrocyte sedimentation rate mmhLactate dehydrogenase ulInterferon group n ““““““““““““““““““Control group n ““““““““““““““““““ 0cH Rahmani et alInternational Immunopharmacology Table Outcomes and complicationsParameter Median IQR or n Time to clinical response medianIQR daysICU admission n Intubation requirementLength of stay in ICU days median IQR daysLength of stay in hospital days median IQR daysAllcause mortality at day Six category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeInterferon group n “““Control group n “ ““pvalueOR95 CI“““shorter [ “ days in the IFN group vs “ days in thecontrol group p ] but length of ICU stay was not significantlydifferent between the groups Allcause 28day mortality was and in the IFN and control groups respectively p Table Safety outcomesA total of and common adverse events were recorded duringthe study period in the IFN and control groups respectively Moreovernumber of serious adverse events was in the IFN group and in thecontrol group The incidence of grade or of adverse events washigher in the control group than the IFN group As it was expected IFNrelated common adverse effects injection site reactions and flulikesyndrome occurred only in the IFN group More patients in the controlgroup experienced ARDS secondary infections septic shock AKI andAHI compared with patients in the IFN group Table Nosocomial infections were detected in patients and patientsin the INF and control groups respectively Bloodstream infection withstaphylococcus aureus was detected in a patient in the INF group Threepatients in the control group experienced ventilator associated pneumonia with klebsiella pneumonia in two patients and acinetobacterbaumannii in another patient Other patients in the control group hadbloodstream infection with staphylococcus aureus DiscussionThis is first randomized clinical trial that evaluated efficacy andsafety of IFN subtype 1b in patients with severe COVID19 In thisstudy Interferon beta1-b as addon therapy significantly shortened the time toclinical response increased the discharge rate at day and decreasedneed for ICU admission in these patients However duration of hospitalization intubation rate length of ICU stay and allcause 28daymortality were not significantly changed Incidence rates of commonand serious adverse events were higher in the control group comparedwith the IFN group The sample size was calculated to assess effect ofInterferon beta1-b on time to clinical improvement in hospitalized patients withCOVID19 However the sample size might not have enough power todifferentiate effects of Interferon beta1-b on the secondary endpointsIFN is a subtype of the type INFs that is released by the lymphocytes as the first cytokine following exposure to viruses It activatesinterferonstimulated genes ISGs after binding to the receptors Theantiviral effects of IFNs are regulated through these genes InadequateIFN response caused uncontrolled viral replication raised viral load andled to poor outcomes in SARSCoV infection A strong IFN responsefollowing infection with SARSCoV2 was detected [“] Expressionof ISGs significantly increased in patients with CoVID19 [] In evaluation of transcriptional responses in various models in vitro ex vivoand in vivo BalancoMelo et al showed that the levels of IFN I andIFN III decreased in SARSCoV2 infection In in vitro model expressions of IFN I and IFN III were not detected in A549 cells as adenocarcinomic human lung cell line infected with SARSCoV2 Of notemoderate increase in the expression of ISGs was observed Next stepthe cells were treated by IFN that caused substantially reduction inthe viral replication Furthermore in ex vivo model the levels of IFN Iand IFN III were undetectable following infection of human bronchialepithelial cells with SARSCoV2 Finally in vivo assessment was considered Postmortem lungtissue samples were extracted from patientswith COVID19 and related transcriptional responses were comparedwith samples from the healthy individuals Similar to previous modelsmodest expressions of ISGs were detected but not about IFNs It is interesting that in all of the models robust cytokine and inflammatoryresponses were noticed []In the study of Yuan et al the antiviral activity of agents including hostbased IFNs IFN 1a Interferon beta1-b pegylated IFN α2a andIFN γ1B and virus targeting antivirals remdesivir and lopinavir wereassessed EC50 of these agents was determined according the plaquereduction assay The most potent IFNs were Interferon beta1-b EC50 IUml and IFN 1a EC50 IUml The EC50 values for remdesivirand lopinavir were determined as and µM respectively TheCC50 values of IFNs remdesivir and lopinavir were IUml µM and µM respectively Among IFNs the most reductive effects on viral load belonged to IFN 1a and Interferon beta1-bHowever Interferon beta1-b showed highest potency and selectivity indexagainst SARSCOV2 []In a randomized clinical trial and patients were recruited in 0cH Rahmani et alInternational Immunopharmacology Fig KaplanMeier plot for estimation of time to clinical improvementthe combination and control groups respectively Patients in the combination group received Interferon beta1-b lopinavirritonavir and ribavirinwhile those in the control group received only lopinavirritonavir Theprimary outcome was defined as the time to reach a negative RTPCR ofrespiratory secretions for SARSCoV2 The time to resolution of thesymptoms was considered as one of the secondary outcomes Themedian time to achieving a negative RTPCR was significantly shorterin the combination group compared to the control group vs days Moreover resolution of the symptoms occurred notably fasterin the combination group than the control group vs days []Similar with our study Interferon beta1-b was started in the viral phase ofCOVID19 ie within first days of onset of the symptoms In our studymedian time from onset of the symptoms to randomization was daysIn both studies first dose of Interferon beta1-b was administered within to h of hospital admission Initiation of antiviral agents as soon aspossible following onset of the symptoms is critical in control of viralreplication and prevention of tissue viral invasion The efficacy of antivirals significantly decreased after establishment of the cytokines release phase in COVID19 [] Due to resource limitations evaluation of viral clearance was not possible in our study No patient died inHung et al study while in our study approximately and ofpatients died in the IFN and control groups respectively Regardingcomparison of the results it should be considered that Hung et alevaluated Interferon beta1-b efficacy in patients with mild to moderate COVID while in our only study patients with severe COVID19 were included Moreover considering severity of the disease incidence rates ofthe serious complications during the hospitalization course were muchhigher in our studyEstebanez et al evaluated the efficacy of Interferon beta1-b in patientswith COVID19 Of them and patients were assigned to the IFNand control groups respectively Inhospital mortality was considered asthe primary outcome of study The mortality rate was statistically significant in the control group than the IFN group vs [] Retrospective design and lack of matching of the groups in termsof receiving other antivirals should be considered when interpreting theresultsIn a case series characteristics and outcomes of five patients withsevere COVID19 who were treated with Interferon beta1-b lopinavirritonavirand hydroxychloroquine were described The antiviral regimen appliedfor these patients was similar to our study Treatment was successful in patients while clinical status of patients deteriorated during thetreatment course All patients received corticosteroids Furthermore allpatients were initially admitted in another hospital and later transferred to the referral hospital [] Clinical outcomes of patients mighthad been affected during lag time of the transfer Moreover patientswere different in terms of the clinical presentations and managementstrategies So definite role of Interferon beta1-b in treatment of these patientscannot be assessedPayandemehr et al evaluated the efficacy of IFN 1a in patientswith moderate to severe COVID19 during a singlearm labelclinical trial All patients received IFN 1a along with hydroxychloroquine lopinavirritonavir and oseltamivir In this study only No at riskInterferon 0Control 0cH Rahmani et alTable Summary of the adverse events during the study periodParameter n Control group n Interferon groupn Any gradeAny gradeGrade or Common adverse events““““““““““““““Grade or “Leukocytosis“Leuk iaLymph ia“Thrombocyt ia“Thrombocytosis“AnemiaHyperkalemia“Hypokalemia“Hyponatremia“Increased creatinine“Increased aspartateaminotransferase“Nausea“Diarrhea“Abdominal pain“Injection site reaction“Flulike syndromeSerious adverse eventsARDS“Nosocomial infection“Septic shock“Acute kidney injury“Acute hepatic injuryARDS acute respiratory distress syndromepatients needed ICU admission and only one death occurred in thehospital Fifteen of the discharged patients were followed for days Noside effects were detected while in our study some patients experiencedcommon adverse effects such as injection site reactions and flulikesyndrome It might be due to receiving concomitant antipyretics andanalgesics that masked these reactions Furthermore main outcomes ofthe study were not welldefined in the method section Duration of thefollowup was only days []The efficacy of IFN 1a in patients with COVID19 was assessed inanother study In this noncontrolled prospective trial patients wereenrolled Five doses of mcg of IFN 1a were administrated subcutaneously on alternate days for these patients The patients also received hydroxychloroquine and lopinavirritonavir for days Theprimary outcome of the study was symptom alleviation during 14dayperiod Within days all patients became afebrile The resolution ofother symptoms gradually occurred [] The oxygenation status andtypes of respiratory supports were not exactly defined In general highflow nasal cannula was applied for most patients and three patientsreceived noninvasive positive pressure ventilation NIPPV No seriousadverse events were detected and none of the patients died Rate of ICUadmission and requirement for invasive mechanical ventilation werenot reported in this study Accounting these limitations absence ofcontrol group and small sample size the interpretation of the resultsshould be done with cautionIn another study efficacy and safety of IFN 1a were evaluated inpatients with severe COVID19 in an label randomized clinicaltrial Fortytwo and patients were recruited to the IFN and controlgroups respectively Time to clinical response based on the six ordinarycategory scale was primary endpoint of this study Following twoweektreatment with IFN 1a time to clinical response was not statisticallydifferent between the groups On day the numbers of dischargedpatients were significantly higher in the IFN group compared with thecontrol group vs Early administration of IFN 1asignificantly reduced the mortality rate compared with late administration [] Absence of followup PCR and chest imaging along withthe small sample size were the major limitations of the studyOur study suffered from some limitations Follow up chest imagingInternational Immunopharmacology or virological assessment was not possible due to resources limitationstherefore the effect of Interferon beta1-b on viral clearance was not determinedSmall sample size did not allow accurate estimation of survival benefitof Interferon beta1-bIn conclusion Interferon beta1-b was effective in shortening the time toclinical improvement without serious adverse events in patients withsevere COVID19 Furthermore ICU admission rate and need for invasive mechanical ventilation significantly reduced by administrationof Interferon beta1-b Although compared with the control group Interferon beta1-b reduced duration of hospitalization length of ICU stay intubation rateand 28day mortality were not statistically different between thegroups Further randomized clinical trials with enough sample size areneeded to accurately estimate survival benefit of Interferon beta1-bCRediT authorship contribution statementHamid Rahmani Data Curation Formal analysis InvestigationWriting original draft Effat DavoudiMonfared Data CurationAnahid Nourian Data Curation Hossein Khalili ConceptualizationMethodology Supervision Writing review editing NooshinHajizadeh Project Administration Narjes zarei Jalalabadi ProjectAdministration Mohammad Reza Fazeli Resources MonirehGhazaeian Resources Mir Saeed Yekaninejad Formal analysisDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paperAcknowledgementWe would like to thank the nurses and other staffs of ImamKhomeini Hospital Complex for their kind supports and also Ms AvaKhalili for English proofreading the manuscriptFundingThe authors did not receive any fund for this workAppendix A Supplementary materialSupplementary data to this article can be found online at 101016jintimp2020106903References[] L Wang Y Wang D Ye Q Liu Review of the novel coronavirus SARSCoV[] JH Beigel KM Tomashek LE Dodd AK Mehta BS Zingman AC Kalil based on current evidence Int J Antimicrob Agents 101016jijantimicag2020105948[] D Cucinotta M Vanelli WHO Declares COVID19 a Pandemic Acta Biomed “ 1023750abmv91i19397[] Johns Hopkins Coronavirus Resource Center Home Page July coronavirusjhuedumaphtml[] JM Sanders ML Monogue TZ Jodlowski JB Cutrell Pharmacologic treatmentsfor coronavirus disease COVID19 a review JAMA 101001jama20206019E Hohmann HY Chu A Luetkemeyer S Kline DL de Castilla RW FinbergK Dierberg V Tapson L Hsieh TF Patterson R Paredes DA SweeneyWR Short G Touloumi DC Lye N Ohmagari MD Oh GM RuizPalaciosT Benfield G Fatkenheuer MG Kortepeter RL Atmar CB Creech J LundgrenAG Babiker S Pett JD Neaton TH Burgess T Bonnett M GreenM Makowski A Osinusi S Nayak HC Lane Remdesivir for the treatment ofCovid19 preliminary report N Engl J Med 101056NEJMoa2007764DK Manson C Kubin RG Barr ME Sobieszczyk NW Schluger Observationalstudy of hydroxychloroquine in hospitalized patients with Covid19 N Engl JMed “ 101056NEJMoa2012410[] J Geleris Y Sun J Platt J Zucker M Baldwin G Hripcsak A Labella[] B Cao Y Wang D Wen W Liu J Wang G Fan L Ruan B Song Y Cai M Wei 0c[] Z Zhou L Ren L Zhang J Zhong Y Xiao Z Jia L Gou J Yang C Wang 101016jantiviral2020104791S Jiang D Yang G Zhang H Li F Chen Y Xu M Chen Z Gao J Yang J DongB Liu X Zhang W Wang K He Q Jin M Li J Wang Heightened innate immuneresponses in the respiratory tract of COVID19 patients

"Data from the Colorado Plateau uranium miners cohort. Interestingly even though model 8 is produced from the flexible definition in (5)“(7) Figures 2 and 3 suggest that the assumption of independency holds here with shapes of the exposure-response and lag-response curves at different values of ?p and xp respectively being proportional and the maximum HR constantly experienced at lag 11. This result reinforces the fact that the cross-basis representation based on a truly bivariate exposure“lag“response function f · w(x?) may appropriately describe the specific independency case defined by the simpler representation f(x) · w(?) in (4). 3.5. Prediction for specific exposure histories The flexible modeling approach described here can be applied to predict the overall cumulative risk from (10) for a specific exposure history qh as outlined in Section 2.3. Table III illustrates the predicted HR from four different models in five alternative exposure scenarios. This approach previously proposed 17 provides clear and interpretable risk summaries from complex models in the presence of varying exposure patterns. The first two scenarios refer to a constant radon exposure of 20 and 100 WLM/year respectively in the past 10 years. As expected simple DLMs (models 1 and 4) predict a similar risk but substantially lower than the two DLNMs with a B-spline for f(x). In particular model 5 extends model 1 by allowing a nonlinear dependency for the unweighted cumulative exposure estimating a slightly lower risk when compared with the more flexible model 8 already described. The third scenario extends the exposure to 20 WLM/year in the previous 20 years while the fourth one assumes that a 10-year exposure to the same intensity ceased 10 years before. The comparative assessment of the four models is similar to the first two examples. The last scenario considers the risk of more remote exposures occurring 30“39 years ago. Interestingly models 1 and 5 provide identical estimates to the fourth scenario as the risk of past exposures is assumed constant along the whole lag period. Model 8 instead predicts no excess in lung cancer in the last scenario given at least 30 years passed from the last exposure to radon a lag period for which the lag“response curve in (bottom-left panel) displays a null risk. Table III Overall cumulative hazard ratio (with 95%CI) of lung cancer mortality associated with alternative scenarios of exposure histories to radon as predicted from models 145 and 8 described in Table II Model 1 Model 4 Model 5 Model 8 Exposure scenario x · c x · w(x?) f(x) · c f · w(x?) 20 WLM/year in the last 10 years 1.05 (1.04“1.06) 1.04 (1.03“1.05) 1.33 (1.22“1.46) 1.52 (1.31“1.76) 100 WLM/year in the last 10 years 1.27 (1.22“1.33) 1.20 (1.13“1.27) 1.96 (1.73“2.22) 2.37 (1.87“2.99) 20 WLM/year in the last 20 years 1.11 (1.09“1.14) 1.11 (1.09“1.14) 1.92 (1.56“2.35) 3.12 (2.29“4.24) 20 WLM/year 10“19 years ago 1.06 (1.05“1.07) 1.07 (1.06“1.09) 1.43 (1.28“1.61) 2.05 (1.70“2.48) 20 WLM/year 30“39 years ago 1.06 (1.05“1.07) 1.05 (1.00“1.11) 1.43 (1.28“1.61) 1.04 (0.64“1.70) WLM working-level months. The summaries illustrated in this section can be extended to predict how the risk evolves dynamically in time in association with time-varying exposures. Adopting a forward perspective the risk changes along an exposure profile with specific exposures events referring to different lags and producing a different exposure history. As an example displays the overall cumulative mortality risk within years 0“60 for an exposure to 20 WLM/year experienced in the first 15 years. Here model 8 predicts an HR peak of 2.94 (95%CI: 2.20“3.93) at around 20 years 5 years after the end of the exposure. The plot also suggests that model 4 assuming a log-linear exposure“response relationship seriously underestimates the risk of lung cancer for four decades predicting an HR at year 20 of 1.11 (95%CI: 1.09“1.13). Also the assumption of a constant risk along lags of a nonlinear relationship adopted in model 5 produces an underestimation of the predicted HR in the first part of the period followed by a clear overestimation in the last years. Trend of hazard ratio (HR) of lung cancer mortality in the period 1“60 years associated with radon exposure of 20 WLM/year experienced in years 1“15 predicted from models 8 (with 95%CI)5 and 4 as specified in Table II. 3.6. On linearity and the ™nonspecial™ case of log transformation The bottom-right panel of reports the exposure“response for the simple DLM in model 4 which predicts a substantially lower risk than the two DLNMs. This difference is also evident when comparing the HR range in Figures 1 and 2 (bottom-left panel). This discrepancy is related to the wrong assumption of a linear radon-mortality dependency with the fit of model 4 highly dependent on a few of very high exposure occurrences. A sensitivity analysis performed on the subset of subjects with a maximum yearly exposure to radon of less than 300 WLM/year (81.6% of the total) is illustrated in Section D.2 of the supporting information. The highly skewed distribution of exposure events to radon and the shape of the estimated exposure“response curve suggest a log transformation of the exposure. In fact this model can be still described as a DLNM in (5)“(7) characterized by a basis with dimension vx = 1 corresponding to f(x) = log(x + 1). A new model is defined by replacing the spline in model 8 with the log function. The comparison is presented in details in Section D.3 of the supporting information. Although this more parsimonious model slightly improves the fit with an AIC of 2148.6 it is worth noting that results are very similar as illustrated in Figure S4 (supporting information) suggesting that the spline function is flexible enough to recover the association. More generally different functions than those presented here can be used to define the exposure“response or lag structure. 4. Simulation study The performance of the extended DLNM framework is validated through simulations under different scenarios of exposure“lag“response associations. Specifically the framework is evaluated by estimating the relative bias coverage and relative root mean square error (RMSE) of the estimators derived from AIC and BIC selection and the empirical rejection rates for the hypotheses H0 : f(x) = x and H0 : w(?) = c of linearity and constant effects respectively. 4.1. Simulation design and data generation The simulation setting involves the generation of exposure profiles for a set of ns subjects the definition of scenarios with known bidimensional exposure“lag“response associations and the random generation of time-to-event occurrences from such scenarios. These steps are briefly summarized here with more detailed information provided in Section E of the supporting information. The time-varying exposure profiles for ns subjects are represented as series of occurrences xt at time t = 1 ¦100 generated by random exposure events with an intensity in the range 0“10. "

" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period “ by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chen™s metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [“] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [“] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes [“]in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the “ period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients™ medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no “ for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at “ weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 ˆ’ t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t ˆ’ t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged “ years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr “ fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr “ delivered in gy dailyfractions iqr “ concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at “ weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range “ months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± “ ± “ ± “the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate œduration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range “ days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range “ and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from ˆ’ to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors “ “ “from days to days apart median days the median tumor volume relative increase rate was perday median range “ and the mediantumor volume doubling time was days range “days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at “ weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range “ monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising [“]many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [“] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate‰¤ vs 1dayvtu volume of primary tumorlocoregional controlhr ci““““pvaluecause specific hazardhr ci““““pvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established [“]however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at “ weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ “] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [“] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [“] in our grouphowever no such association was found a small numberof patients and “ in contrast to other studies “ the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at “ weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [“ “] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers [“]sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be œasshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors™ contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3“availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no “ all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ world health anization who life expecta

"peroxisome proliferatoractivated receptorsppars asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms pparalpha ppar betadelta and ppar gamma ppars formheterodimers with retinoic x receptors and regulate theexpression of various genes upon ligand binding ppars alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes ppars asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions for instance ppar alpha also functions in fatty acidbetaoxidation and vascular ‚ammation ppar gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes ppar betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression ppar alpha and ppar gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] on the contraryppar betadelta produces more obviously proangiogeniceï¬ects [“] in this review we will focus on the promotingrole of ppar betadelta in angiogenesis especially in tumorangiogenesis the network of interplay between ppar betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct ppar betadelta target genes angiogenesisangiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells epc migrate to sites of vascularization then diï¬erentiate into endothelial cells ec andcoalesce into the initial vascular plexus [ ] besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cppar researchmmps extracellular matrix ecm and basement membrane componentsangiogenesis is a physiological and vital process in development and growth an imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growththus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] inducing angiogenesis is known as a hallmarkof cancer and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones tumor angiogenesis tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis the tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningtherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread thus this has been regarded as a critical target forantitumor therapy ppar alpha and angiogenesisit was reported firstly that a selective ppar alpha agonistwy14643 did not show any eï¬ect on angiogenesis or ec proliferation but some subsequent studies showed that theactivation of ppar alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used ppar alpha agonist moreover fenofibrate suppressed ec proliferation migration and tube formation through inhibition of protein kinaseb akt and disruption of the cytoskeleton furthermore ppar alpha activation was shown to inhibit vascularendothelial growth factor vegf induced ec migrationand basic fibroblast growth factor bfgffgf2 inducedcorneal angiogenesis in vitro and in vivo especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma lewis lungcarcinoma llc fibrosarcoma and glioblastoma due to asystemic treatment of ppar alpha ligand and the antiangiogenic state induced through activation of ppar alpha withelevated thrombospondin1 tsp1 and endostatin expression howeverit was demonstrated inanother observation that activation of ppar alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase enos and akt via avegfdependent manner furthermore zhang andward also suggested that ppar alpha activation inducedproangiogenic responses in human ocular cells inanother study it was shown that a new ppar alpha agonistrk13675 had no eï¬ect on angiogenesis recentlyppar alpha activation is further shown to have antineovascularization eï¬ects with downregulation of vegf and angiopoietin expression in a rat alkali burn model in summary the role of ppar alpha in angiogenesis isstill controversial some observations showed that ligandactivation of ppar alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as tsp1 and endostatin or downregulation of proangiogenic factors including vegf fgf2 akt and angiopoietins others also reported opposite results showing aproangiogenic role upon ppar alpha activation thus thespecific molecular mechanism is still unclear and needs tobe further studied ppar gamma and angiogenesisligand activation of ppar gamma was previously shown toinhibit human umbilical vein endothelial cell huvec tubeformation in collagen gels and vegfinduced choroidalneovascularization in vitro and in vivo another studyalso demonstrated that ec apoptosis was induced throughtreatment with the ppar gamma ligand 15dpgj2 furthermore rosiglitazone a potent ppar gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bfgfinduced corneal neovascularizationin vivo moreover a similar observation also displayedthe inhibition of vegfinduced angiogenesis in a chickchorioallantonic membrane model in a mouse modelwith ischemiainduced retinopathy pioglitazone a ppargamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin additionally theppar gamma antagonist gw9662 was shown to reverseomega3 polyunsaturated fatty acidinduced reduction ofeselectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 implicatingan antiangiogenic potential of ppar gamma itself howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of epc in vitro andin vivo via a phosphoinositide3kinase pi3k dependentmanner nadra observed that ppar gammanull embryosdisplayed a vascular structural defect at e95 moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the ppar gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingvegf proliferin and plateletendothelial cell adhesionmolecule1 pecam1cd31 suggesting a crucial roleof ppar gamma in placental vascular development the 0cppar researchmajor antiangiogenic properties on ppar gamma activationwere also reviewed here notablyin most cancersthe canonical wntbetacatenin pathway is upregulated while on the contrary ppargamma is downregulated interestingly in numerous tissuesthe activation of ppar gamma inhibits the betacateninpathway whereascanonicalwntbetacatenin signal cascade also inactivates ppargamma implicating a negative regulatory role of ppargamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthethein summary ppar gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of vegf or bfgfinduced neovascularization andreduction of the expression level of some proangiogenicfactors ppar betadelta and angiogenesisunlike ppar alpha and ppar gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof ppar betadelta on physiological and pathological angiogenesis the first evidence provided in a study is that activation of ppar betadelta with gw501516 a highly selectiveppar betadelta agonistinduces huvec proliferationand an increased expression of vegf and its receptorvegfr1 flt1 besides inducing ec proliferationppar betadelta activation by itsligand prostacyclinpgi2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ecs from h2o2induced apoptosis and oxidant injury moreover a subsequent study further provides evidence that activation of ppar betadelta withgw501516 induces angiogenesis during which vegfrelease is considered as a major trigger factor firstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice moreover reexpression ofppar betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization indicating a crucial role of ppar betadelta in tumorvascularization additionally another study also observedreduced levels of calcium intracellular channel protein clic4 but it observed enhanced expression of cellular retinol binding protein crbp1 in migrating ecs from pparbetadeltanull mice both of which play a role in tumorvascularization [ ] it was reported that ppar betadeltawas required for placentation and most of the pparbetadeltanull mutant embryos died at e95 to e105 due toabnormal celltocell communication atthe placentaldecidual interface however in these studies [“] adefect in angiogenesis was not observed during normal development in ppar betadeltaknockout micesome observations also show the important role of pparbetadelta in physiological angiogenesis for instance skeletal musclespecific ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] moreover pparbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective ppar betadelta agonistgw0742treated animals furthermore in the heartpharmacologicalstimulation withgw0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin interestingly the same cardiac phenotype wasalso observed after treatment with the ppar betadelta agonist gw501516implicating a response specificity forppar betadelta stimulation calcineurin activationfurther leads to the stimulation of nuclear factoractivatedt cell c3 nfatc3 and an enhanced expression of hypoxiainducible factor alpha hif1alpha and cyclindependentkinase cdk9 overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinppar betadelta may act as a key regulator in mediatingpathological angiogenesis for instance ppar betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an angiopoietinlike protein angptl4 dependent manner implicating the potential of pparbetadelta in modulating pathological ocular angiogenesisrecently an observation reported that ppar betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andppar betadelta promoted laserinduced choroidal neovascular cnv lesions in ppar betadelta mice moreover pharmacological inhibition of ppar betadelta with theantagonist gsk0660 also resulted in a significantly decreasedcnv lesion size in vivo suggesting a functional role of pparbetadelta in the development of cnv lesions this indicates that ppar betadelta has an important association withpathological angiogenesisangiotensin ii ang ii the biologically active peptide ofthe reninangiotensin system ras is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction inexperimental systemsthe antitumor eï¬ects of diverseace inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] it has been shown recentlythat activation of ppar betadelta inhibits ang iistimulated protein synthesis in a concentrationdependentmanner and suppresses ang iiinduced generation of reactive oxygen species ros in vascular smooth muscle cells ppar betadelta was further shown to inhibit angiimediated atherosclerosis however it is not clearuntil now if ppar betadelta activation can be consideredis foras an ace inhibitormimicking approach as itexample the case for ppar gamma activators antiangiogenicantimetastatic 0cppar researchthe relevance offurthermorethis hypothetical pparbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologybesides inducing angiogenesis it has been demonstratedthat ppar betadelta directly acts on early epc through activation of the akt pathway and induces an enhanced vasculogenesis similarlythe ppar betadeltamediatedprovasculogenic eï¬ects are also observed on late epc he showed that ppar betadelta activation withgw501516 induced epc proliferation and tube formationwhereas epc treated with an inhibitor of cyclooxygenasecox or pgi2 synthase or with ppar betadeltaspecificsirna also displayed an opposite eï¬ect furthermoreit has been demonstrated that ppar betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase mmp 9mediated insulinlikegrowth factor1 paracrine networks upon epc activation han also observed that ppar betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound overallin addition to ec ppar betadelta is also a key regulator ofepc or even may act as an initiator of activation of epc tofurther induce vasculogenesis ppar betadelta and tumor angiogenesislinked to tumor microenvironmentppar betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of ppar betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesthus here we discuss the promotion of ppar betadelta intumor progression through facilitating tumor angiogenesisppar betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] in the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in ppar betadeltanullmice in comparison with wildtype mice moreoverthe elevated ppar betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer indicating the crucial association of ppar betadelta withtumor angiogenesis progression and cancer invasivenessppar betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment tme where tumor angiogenesis is fostered moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withtme to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression for instance it wasshown that colon cancer cells with ppar betadelta knockoutfailed to stimulate ec vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that ppar betadelta is required for the promotion of angiogenesis in hypoxic stressmediated tme moreover in the tme tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites interestingly it hasbeen demonstrated that ppar betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an interleukin il10 dependent manner moreover impairedtumor growth and angiogenesis were observed in pparbetadelta ko bmt mice due to ppar betadelta deficiencyin tumor myeloid cells suggesting that ppar betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of tmefurthermore the endoplasmic reticulum er an essential anelle involved in many cellular functions is implicated in tme in cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt er function and lead toaccumulation of unfolded proteins in er a condition knownas er stress cells adapt to er stress by activating an integrated signal transduction pathway called the unfolded protein response upr upr represents a survival response bythe cells to restore er homeostasis and has both survivaland cell death eï¬ects the mechanisms that determine cellfate during er stress are not well understood for instanceshort exposure to er stress initially increases akt signalingbut longterm er stress suppresses akt signaling ppar betadelta activation has been shown to reduce endoplasmic reticulum er stressassociated ‚ammation inskeletal muscle through an ampkdependent mechanism and to reduce ‚ammation in response to chronic erstress in cardiac cells furthermore it has been nicelyshown that ppar betadelta can repress rasoncogeneinduced er stress to promote senescence in tumors thisis mediated through the decrease of pakt activity promoting cellular senescence through upregulation of p53 and p27expression it would be interesting to investigate thedirect eï¬ects of ppar betadelta on senescence of tumorendothelial cells in an in vivo setting we recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions if ppar betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon ppar betadelta activationobserved by us and others [ ]most recently zuo demonstrated that pparbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including vegf and interleukin il8 most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspecificppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in specific tumor or cancer cell types wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] crosstalk between ppar betadelta and signalmolecules ppar betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as vegf pdgf and fgfproinvasive matrixdegrading enzymes such as mmp9pro‚ammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identified as ppar betadelta directtarget genes besides a leading role of ppar betadelta amongthe signal molecules ppar betadelta may function in tmelinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules interplay between ppar betadelta and ‚ammatoryangiogenesis ‚ammatory angiogenesis is a crucial processin tumor progression for instance the pro‚ammatorymediator cyclooxygenase2 cox2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of vegf and induction of mmps moreover selectiveinhibition of cox2 has also been shown to suppress angiogenesis in vivo and in vitro it is well known that vegfaplays a critical role in both angiogenesis and vasculogenesis and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] ithas also been demonstrated that mmp9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of vegf to its receptors furthermore it hasbeen reported that ‚ammatory cell mmp9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingmmp9 leptin is shown to mediate angiogenesisin vivo and in vitro through induction of ec proliferationand expression of mmp2 and mmp9 and to furtherpromote ec diï¬erentiation and directional migrationthrough enhancement of cox2 activity leptin couldalso induce angiogenesis via transactivation of vegfr inecs additionally besides inducing angiogenesisppar betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of cox2 andits product prostaglandin e2 pge2 in vivo [ ]interestingly cox2 vegf mmp9 and leptin have beenidentified as ppar betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells colorectal cancer cells [ ] epcs[ ] and liposarcoma cells respectivelylinks between vegf and mmpsin tme tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive mmp9and ‚ammatory chemokines [“] chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ecs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis chemotaxis of tumor cells and stromal cells in tmeis also required for tumor dissemination during tumor progression and metastasis [ ]cxc chemokines such as cxcl8 encoding il8 andcxcl5 are also involved in cox2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] it is further shown that il8 directly regulatesangiogenesis via recruitment of neutrophils whichfurther drives vegf activation moreoveril8responding neutrophils are considered as the major sourceof angiogenesisinducing mmp9 [ ] chemokine cc motif ligand ccl2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis furthermore myeloid monocytic cellssuch asmyeloidderivedtumormdscsassociated macrophages tams and dendritic cells arerecruited to the tumor site mainly by ccl2 and producemany proangiogenic factorssuch as vegf cxcl8plateletderived growth factor pdgf and transforminggrowth factor beta tgf beta [“] in fact bothtgf beta and hypoxia are potentinducers of vegfexpression in tumor cells and collaborate with tme toprovide the foundation of tumor angiogenesis and cancercell invasion importantly il8 has been reported asa key target gene of ppar betadelta to promote angiogenesis in vivo and in vitro and ccl2 expression isalso significantly upregulated upon vascular ppar betadelta overexpression in vivo suppressorcellscox2 also mediates il1 betainduced angiogenesisin vitro and in vivo [ ] il1 beta supports neovascularization through the regulation of the expression of vegfand its receptor vegfr2 flk1kdr on ecs il1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ecs and leukocytes which facilitates tumorassociated angiogenesis in tme ‚ammatory il1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as vegf vegffurther activates ecs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis in addition il6 also stimulates angiogenesis and vasculogenesis[ ] however gopinathan observed an il6induced newly forming vascular structure with defectivepericyte pc coverage ex vivo thus facilitating cancercell ltration and tumor metastasis through vascular leakage interestingly il1 and il6 expression levels are significantly upregulated in the ppar betadelta overexpressionmouse model reported recently in summary ppar betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto tme in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating myeloid cells such as neutrophils tams and mdscs other key ppar betadeltamediated proangiogenicfactors it has been demonstrated that wilms™ tumor suppressor wt1 is a major regulator of tumor neovascularization andtumor progression e26 avian leukemia oncogene ets1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis in addition ets1 promotes cancer cell invasion throughupregulation of mmps consistent with this silencingof ets1 in highly invasive breast cancer cells also reducesthe expression of mmp9 and mmp1 ets1 also acts as a key regulator of mmps such asmmp1 mmp3 and mmp9 in human cancerassociatedfibroblasts cafs [ ] cafs support tumor growthby secreting growth factors such as vegf fgf pdgf andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]cafs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ecmstructure thus helping a tumor to acquire an aggressive phenotype [ ] ppar betadelta in cafs also exhibits aprotumorigenic eï¬ect it was reported that ablation of pparbetadelta in cafs attenuated tumor growth by altering theredox balance in tme suggesting that ppar betadeltain cafs is also an important player in tumor developmentets1 induces the expression of vegf vegfr1 andvegfr2 in ecs [“] in turn vegf is also a majorinducer of ets1 in ecs through the activation of either thepi3kakt pathway or the mekerk12 signal cascade[ ] wt1 is also reported to regulate tumor angiogenesis via direct transactivation of ets1 sryrelated hmgbox sox18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing and cancer progression and most recently it was further shown that specificecderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of sox18 interestingly theseimportant proangiogenic molecules including wt1 ets1and sox18 are also significantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identified as a target gene of ppar betadeltain melanoma cells ppar betadelta may facilitate cancer progression atdiverse cellular levels in tme ppar betadelta activationis shown to induce colonic cancer stem cell csc expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the nanog gene nanog as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells and cancer cellsexpressing nanog also often exhibit stem cell properties protooncogene ckitcd117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in cscs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors moreover ckit has been identified as a potentialmarker of the cancer stemlike cells in addition ckitnot only functions on ecs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] studiesalso suggested that activation of ckit enhances the expression of vegf that can be suppressed by imatinib an inhibitor of ckit in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] ckit is also involved in pathological ocular neovascularization and is regulated transcriptionally by wt1 and ppar betadelta pdgfb and its receptor pdgfr beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ecs [“] andepcs and to regulate vascular permeability and vesselmaturation through recruitment of pericytes pcs and smooth muscle cells smcs in newly formingvessels moreover pdgfb and pdgfr beta also interactwith other proangiogenic factors such as fgf2 [ ]vegfa and its receptor vegfr2 furthermorepdgfb and pdgfr beta may also aï¬ect cancer growthand progression by directly acting on tme besides thecrosstalk with cafs [“] pdgfr beta in stromalfibroblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that specific targeting of pdgfr beta kinaseactivity in tme inhibited cancer growth and vascularizationin cancers with high pdgfb expression such as llc therefore this indicates the diverse role of pdgfb andpdgfr beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in tme pdgfr beta is demonstrated as a target of telomeric repeat binding factor trf2 that is further activated transcriptionally by wt1 pdgfb and pdgfr beta have further been identifiedas critical targets of ppar betadelta via a direct transactivation mechanism in vivo in conclusion a variety of key signal molecules involvedin tumor angiogenesis and tumor progression and metastasishave either been identified as ppar betadelta direct targetsor largely upregulated in the vascular ppar betadelta overexpression model in vivo reported recently thus pparbetadelta activation seems to give rise to a highly angiogenicphenotype and even plays a œhallmark role in promotingtumor angiogenesis and progression interestingly it appearsthat there could also exist a widely interactive networkbetween the downstream protumorangiogenic moleculesas described above therefore the crosstalk network is established between ppar betadelta and the various signal molecules and also between those molecules figure 1amoreover in addition to cancer cells ppar betadeltamay also produce pleiotropic eï¬ects in tme by modulatingdownstream key molecules to act on ecs epcs pcs smcscscs cafs and tumorltrating ‚ammatory cells indirectly facilitating tumor angiogenesis and further promotingcancer development figure 1b other ppar betadelta target genes ppar betadeltaregulates the transcription oftarget genes via a direct 0cppar researchil1 betacox2leptinppar betadeltavegfvegfrpdgfr betatrf2wt1ets1mmp9pdgfbckitcxcl8il8il10ccl2cxcl8il8pdgfr betaappar betadeltaets1mmp9pdgfr betananogckittumorassociatedmacrophage tamdendritic cellneutrophilmyeloidderivedsuppressor cell mdscvegfmmp9pdgfbckitsox18pdgfr betaendothelial cell ece

"Identifiability and constraints The tensor product structure of the cross-basis defined in (5)“(7) poses some identifiability issues. In particular each of the vx basis variables in R is multiplied by each of the v? basis variables in C. If an intercept is included in f(x) the related matrix of cross-basis variables W is not of full rank and the parameters of the regression model are not identifiable even when a common intercept is not included. Therefore the cross-basis in (7) should always be defined without an intercept in the basis functions for x. Also these basis functions can be centered on a specific exposure value x0 which will represent the reference for the risk summaries computed by (8)“(10). The bidimensional shape of the exposure“lag“response can be constrained to follow a prespecified pattern. In particular a priori assumptions on the lag structure can be imposed through functional constraints on the basis for the space of ?. Left and right constraints on the extremes of the supporting interval ?0“L are particularly meaningful for smooth functions. A left constraint can be imposed by excluding the intercept from the basis. This step will force the lag“response curve to predict a null risk at the beginning of the lag period. A right constraint on a B-splines basis can be produced by excluding specific basis variables as previously described for linear exposure“response relationships 17. The constraint produces a smooth dependency which approaches a null risk at the end of the lag period. Such constraints are particularly useful in the presence of sparse data in order to limit the flexibility of the model under specific assumptions about the lag“response curve. However biases can be introduced if these assumptions are not met. Additional information is provided in Section D1 of the supporting information. The functional constraints discussed in this section can be specified without introducing customized optimization methods for estimating the parameters ? in (3)“(7). More sophisticated methods are required for example to constrain the lag“response curve to be non-negative in the whole lag period L. These approaches have been previously proposed for linear dependencies 141718 and introduce further complexities in the bidimensional context of DLNMs. This development is not pursued here. 2.5. Model selection and inferential procedures The framework described in Sections 2.1“2.2 includes a fairly large number of models defined by different functions for each of the two dimensions and by different choices regarding each function such as number and location of knots in splines. This raises the issue of selecting the optimal model for describing the exposure“lag“response association. Previous studies on temporal dependencies have proposed selection procedures on the basis of profile likelihood 15 AIC 141620 or BIC 17. Simulation studies seems to indicate a better performance of AIC when compared with BIC in this context 18 a result consistent with unpublished simulations performed on time series data for DLNMs. Inference on the models illustrated in the previous sections primarily focuses on the specification of confidence intervals for the risk measures in Section 2.3 and on the definition of tests for a set of null hypotheses. Confidence intervals for lag“response curves exposure“response curves and cumulative risks obtained through and can be easily derived from the diagonal of the related (co)variance matrices in (8)“(10) assuming a multivariate normal distribution of the estimators. Regarding hypothesis testing two null hypotheses are particularly relevant in this framework. The first one postulates a linear exposure“response relationship namely H0 : f(x) = x. The second one assumes a constant risk namely H0 : w(?) = c. Tests on constrained models can be also defined. The assumption of independency is not easily tested as the form in (4) cannot be expressed as a model linear in its parameters. However defining general inferential procedures in this setting is not straightforward. First the null hypotheses H0 : f(x) = x and H0 : w(?) = c are not independent and an incorrect assumption about the association in one dimension may bias the test estimator for the hypothesis related to the other space as previously reported 19. In addition estimates are usually conditional on a posteriori selection of a best-fitting model based on the selection methods discussed before. Under these conditions the estimators for the (co)variance matrices in (8)“(10) are likely to underestimate the true sampling (co)variance and the distribution of the test statistics may be different from that assumed unconditional on the selection procedure. This may generate undercoverage of confidence intervals and inflated type I error for tests 1727. Given these complexities a general framework for hypothesis testing embedded in the model selection procedure is not provided here. An assessment through simulations of the performance of estimators generated by AIC and BIC-selected models will be presented in. Specifically simulations will provide an empirical evaluation of the ability of the information criteria to identify the correct model between those defining the null or alternative hypotheses about linearity and constant effects and measures of performance such as bias coverage and root mean square error. 3. An application The conceptual and statistical framework of DLNMs described in extended beyond time series data is general and applicable in different study designs. As an illustrative example I propose here an application in survival analysis of time-to-event data. This represents one of the most complex settings as the temporal pattern of risk is produced by exposure histories that vary during the follow-up of each subject. Specifically the methodology is used to investigate the association between occupational exposure to radon and mortality for lung cancer. The analysis is based on data from the Colorado Plateau uranium miners cohort already used in previous methodological contributions 121520. Section A of the supporting information provides a list of the main steps to replicate the analysis in other real-life examples. 3.1. Data The cohort data used in this example were collected by the National Institute for Occupational Safety and Health. Detailed information on the cohort is given elsewhere 12. Briefly subjects were eligible to enter the cohort if they worked in mines within the Colorado Plateau area between 1950 and 1960 and provided demographic personal and occupational information during their working period. Vital status and cause of death were ascertained by linkage with different sources. The data used in this example refer to the follow-up of the cohort on December 311982 including 3347 subjects and 258 lung cancer deaths. Exposure data available in the data set include cumulative measures of radon and smoking in 5-year age intervals. The radon exposure history for each subject expressed in working-level months (WLM) was reconstructed by linking employment information with measured or predicted levels in each mine in each year. The smoking history expressed in the number of cigarettes packs — 100 was reported by each subject during his working period and assumed constant after the last reporting age. A summary of the data is provided in Table I. Table I Descriptive statistics of the Colorado Plateau uranium miners cohort. The data included here refer to the follow-up on December 31 1982. Exposure to radon is measured in working level months (WLM) while smoking is reported as packs of cigarettes/100 Full cohort Lung cancer cases N % N % Subjects 3347 100.0 258 7.7 Deaths (%) 1258 37.6 258 100.0 Ever smokers (%) 2656 79.4 238 92.2 Median Min 25th 75th Max Median Min 25th 75th Max Age at entry 34.0 15.8 25.8 44.0 80.0 41.6 18.6 34.3 48.0 63.9 Follow-up time (years) 23.9 0.1 19.6 25.5 32.5 18.3 0.3 12.9 22.0 30.8 Exposure to radon Exposure period (years) 6.7 0.1 2.7 11.8 53.0 12.8 0.1 7.8 17.6 39.5 Total cumulative exposure (WLM/year) 429.0 0.0 153.5 1016.8 10000.0 1231.9 8.0 553.7 2528.6 10000.0 Yearly exposure (WLM/year) All 60.2 0.1 26.7 122.2 3245.3 81.6 1.0 42.3 165.4 1295.7 Lag 0“9 52.4 0.1 23.8 102.5 2994.0 61.4 3.9 31.3 144.7 1110.8 Lag 10“19 53.8 0.1 24.3 112.5 3245.3 78.3 1.0 42.9 164.0 1295.7 Lag 20“29 74.0 0.1 33.0 141.7 3245.3 104.7 4.1 52.2 180.0 1295.7 Lag 30“40 95.7 0.2 48.0 151.6 2994.0 104.7 5.5 60.0 175.3 860.2 Smoking Exposure period (years) 38.0 5.0 31.0 46.0 75.0 40.0 14.0 33.0 48.0 72.0 Total cumulative exposure (packs — 100) 131.6 0.4 94.5 174.5 676.3 147.4 21.8 109.5 188.1 567.2 Yearly exposure (packs — 100) 3.6 0.0 2.5 3.6 24.4 3.6 0.0 3.5 4.2 13.4 3.2. Modeling strategy For this illustrative example the analysis is performed through a Cox proportional-hazard model with time-varying covariates by using age as the time axis. Effect measures are reported as a hazard ratio (HR). The model is represented by the following: (11) where the log-hazard log [h(t)] is expressed as a sum of baseline log-hazard log [h0(t)] and contributions of additional covariates. These comprise cross-basis functions sx(xt) and sz(zt) for radon and smoking respectively as defined in (1)“(7) and a linear term for calendar time u in order to control for secular trends in lung cancer risk not accounted for by the delayed effects of the two exposures."

" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim

"Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%). We successfully determined protein levels in all 85 patients. Ten of the 81 eligible patients did not achieve assignment to treatment versus observation within the 84-day time interval from surgical resection. The time interval from surgery to assignment ranged from 86 days to 105 days in these 10 patients. For 3 patients the specimens were received after the 84-day limit had passed. For the other 7 patients the time interval from receipt to reporting ranged from 7 days to 25 days (median 18 days). For the 71 patients with a successful assignment within the 84-day time interval from surgical resection the time from receipt to reporting ranged from 3 days to 26 days (median 8 days). The reasons for reporting results in excess of 14 days were equipment failure and inadequate expression values in control specimens which required equipment recalibration and a repeat processing of the specimens. Overall the time from receipt of specimens to reporting ranged from 1 day to 27 days (median 11 days; mean 12 days) which is similar to that reported for patients with advanced NSCLC (range 1 day-47 days; median 11 days; mean 12 days).18 Survival and Toxicity Survival analyses were performed on the 61 patients who accepted assignment to treatment (44 patients) or surveillance (17 patients). Patients who rejected their treatment assignment withdrew consent and thus could not be followed for survival. Fourteen patients had DFS events; 2 had died (1 from disease recurrence and the other from cardiac disease without recurrence). The median follow-up among those patients still alive at the time of last follow-up was 27 months (range 3 months-44 months). Six patients had <?24 months of follow-up. The collective 2-year DFS and OS rates were 80% (95% confidence interval [95% CI] 67%-88%) (Fig. 2A) and 96% (95% CI 87%-99%) from the date of registration. The 2-year DFS rate was 83% (95% CI 68%-92%) for patients who received chemotherapy (Fig. 2B) and it was 71% (95% CI 43%-87%) for those observed (Fig. 2C). includes 2-year DFS estimates within each of the 3 gene expression categories in the chemotherapy arm. The median time from surgery to enrollment was 41 days (range 11 days-79 days). The time from surgery was added as a covariate to a Cox regression model and was not found to be significantly related to DFS (P?=?.22) or OS (P?=?.36). Disease-Free Survival Rates Patient Group No. DFS (95% CI) 1-Year 2-Year Accepted assigned treatment 61 88% (77%-94%) 80% (67%-88%) Received chemotherapy 44 95% (83%-99%) 83% (68%-92%) By protein level category (for those that received chemotherapy) ?Low ERCC1/low RRM1 20 95% (69%-99%) 84% (59%-95%) ?Low ERCC1/high RRM1 18 94% (65%-99%) 82% (55%-94%) ?High ERCC1/low RRM1 6 100% (100%-100%) 100% (100%-100%) Abbreviations: 95% CI 95% confidence interval; DFS disease-free survival; ERCC1 excision repair cross-complementing group 1; RRM1 ribonucleotide reductase M1. Kaplan-Meier survival estimates are shown. (A) Collective disease-free survival is shown for patients who accepted adjuvant chemotherapy or observation based on gene expression analysis. (B) Disease-free survival is shown for patients who received adjuvant chemotherapy. (C) Disease-free survival is shown for patients in the observation group. Conf Int indicates confidence interval. A total of 22 patients discontinued chemotherapy because of treatment-related toxicity (50%). None of the patients died because of treatment-related toxicity. Details are provided in . Number of Patients With Grade 3 and Grade 4 Adverse Events Among the 44 Patients Who Received Chemotherapya Level of Severity Adverse Event Grade 3 Grade 4 No. of patients with events 13 14 Type of events ?Neutropenia 11 6 ?Thrombocytopenia 4 4 ?Nausea 4 0 ?Vomiting 4 0 ?Anemia 2 0 ?Anorexia 2 0 ?Fatigue 2 0 ?Febrile neutropenia 1 1 ?Thromboembolism 1 1 ?Dehydration 1 0 ?Hearing impairment 1 0 ?Mucositis 1 0 ?Pleural effusion 1 0 ?Renal failure 1 0 ?Bradycardia (sinus) 1 0 ?Syncope 1 0 ?ALT elevation 1 0 ?Hypokalemia 1 0 ?Hyponatremia 0 2 Abbreviation: ALT alanine aminotransferase. a Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (version 3.0). In Situ ERCC1 and RRM1 Protein Levels RRM1 levels ranged from 2.4 to 234.3 (median 39.7; mean 48.1) which were not significantly different from the expected values (median 40.5; range 8.3-96.2) (P?=?.87).16 ERCC1 protein levels ranged from 4.3 to 211.2 (median 41.9; mean 58.8) and these values were significantly different from the expected values (median 65.9; range 1.9-178.7) (P?=? 0.02). There was a significant correlation noted between ERCC1 and RRM1 levels (correlation coefficient 0.39; P?=?.0003) (Fig. 3) as previously reported.91618 Distribution of excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) levels in eligible patients is shown. The median protein levels of ERCC1 in adenocarcinomas squamous cell carcinomas and the other histologies were 34.257.1 and 121.5 respectively. The corresponding median levels of RRM1 were 38.142.6 and 48.9 respectively. Although the levels were higher in squamous cell carcinomas compared with adenocarcinomas the medians were not statistically significant (ERCC1: P?=?.16; RRM1: P?=?.72). DISCUSSION Disease stage is a predictor of benefit from adjuvant chemotherapy in patients with NSCLC. Patients with stage III disease derive the most benefit and those with stage I are reported to derive the least.12419“23 Although not statistically significant for patients with stage I disease and a tumor diameter >?3 cm a numerical risk reduction of 7% has been reported and for those with tumors measuring ??3 cm a numerical risk increase of 40% has been reported.23 A significant treatment-related toxicity is febrile neutropenia which has been reported in 7% to 24% of patients.242022 Treatment-related deaths occur in 0.5% to 2% of patients.122022 The inclusion of molecular markers predictive of therapeutic efficacy into adjuvant decision algorithms would greatly improve the clinical benefit and reduce toxicity for patients with NSCLC. This approach is particularly attractive for patients with stage I disease in whom the parameters for weighing risks and benefits are to our knowledge the least well defined. Recent advances in molecular diagnostics have resulted in improved outcomes for patients whose tumors harbor mutations in oncogenic signal transduction molecules that can be inactivated by therapeutic agents. Similarly platinum agents target DNA and gemcitabine targets ribonucleotide reductase; both are unequivocally required not only for cellular proliferation but also for other essential cellular functions. Although to our knowledge specific oncogenic mutations have not been identified to date ERCC1 and RRM1 have emerged as promising predictors of efficacy for cisplatin and gemcitabine respectively. We conducted a phase 2 trial of treatment selection based on the levels of protein expression of ERCC1 and RRM1 for patients with completely resected stage I NSCLC and tumor diameters ??2 cm primarily to establish feasibility but also to evaluate preliminary efficacy as assessed by 2-year survival rates. We achieved our primary goal by demonstrating within a cooperative group environment that treatment assignment can be achieved for >?85% of patients within 84 days (12 weeks) the established timeframe for the initiation of adjuvant therapy from surgery in patients with NSCLC.12420“22 At first glance our demonstration of feasibility should not be surprising. However it is important to note that surgical practice has not usually engaged a medical oncologist at the time of initial therapeutic planning but rather after complete recovery which substantially reduces the time available for molecular testing before the initiation of adjuvant treatment. We found no difference (P?=?.20) between academic and community sites in the time elapsed from surgery to the receipt of specimens in the reference laboratory (community sites: 57 patients; median 48 days [range 18 days-90 days]; academic sites: 24 patients; median 53 days [range 20 days-90 days]). The time elapsed from specimen receipt to reporting (median 12 days; range 1 day-27 days) was similar to our previous experience in an international trial of patients with advanced NSCLC (median 11 days; range 1 day-47 days).18 Based on these observations we conclude that the current process for routine specimen procurement handling and shipping to a reference laboratory requires substantial improvements to facilitate implementation of molecularly based therapeutic decision-making. For example a developing National Cancer Institute-sponsored project Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trial (ALCHEMIST) which will randomize patients with epidermal growth factor receptor-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC to targeted therapy or not will need to carefully consider these logistical issues. Prior results from adjuvant trials and a retrospective staging project in patients with stage I disease after complete surgical resection have reported 2-year DFS rates of 72% to 74%20 and rates of 68% to 75% for patients with stage IB disease.4 The corresponding 2-year OS rates were 80% to 88% for patients with stage I disease2024 65% to 90% for patients with stage IB disease242225 and 85% for those with stage IA disease.25 Thus our results of a 2-year DFS rate of 80% and OS rate of 96% appear favorable by comparison. However it is prudent to be cautious because we lost 20 of 81 patients from the survival analysis because of consent withdrawal and a direct comparison of outcomes data among trials cannot account for differences in study populations eligibility and staging criteria and provisions for data collection and analysis. The spectrum of protein levels for ERCC1 and RRM1 significant correlation of levels between both molecules and distribution of patients into the 4 gene expression categories in the current study is consistent with previous experience.91213161826 However the current analysis method for biomarker evaluation (ie antibody-based assessment of in situ protein levels) is not suitable for general clinical implementation for several reasons. First ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents and only 1 isoform appears to be involved in platinum-induced DNA damage repair.27 Second the monoclonal antibody 8F1 which is consistently used for ERCC1 protein expression analysis detects a second and unrelated protein that shares a common epitope with ERCC1.28“30 This observation may account for the highly batch-dependent performance of this antibody1827 which may explain the significantly lower ERCC1 values in the current study compared with prior results.16 Third protein levels for RRM1 in particular and to a lesser degree for ERCC1 appear to be influenced by the specimen processing and handling procedures used at collection sites.26 Finally although the method for immunofluorescence-based quantitative detection of both molecules performs well if all specimens to be analyzed are processed simultaneously there is considerable interassay variability if specimens need to be processed individually over an extended period of time as required for real-time patient decision-making.18 However it is important to note that the biochemical biophysical and cell biological evidence for ERCC1 and RRM1 as predictive molecules for platinum and gemcitabine efficacy remains undisputed.510“12273132 A small number of recent clinical trials have used ERCC1 prospectively for therapeutic decision-making. These include 2 randomized phase 3 trials in patients with advanced-stage NSCLC (1 published [NCT00499109]18 and the other terminated and unpublished [NCT00801736]) and 2 adjuvant trials 1 of which was a terminated and not yet published phase 2 trial [TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE) NCT00775385] and the other an ongoing phase 3 trial [International TAilored Chemotherapy Adjuvant trial (ITACA); EudraCT 2008-001764-36]. Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however the authors raised the possibility of a false-negative result because of an inexplicably divergent survival in an internal control group.18 The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms27 and specificity problems with the 8F1 antibody.28“30 The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin-based control treatment with OS as the primary endpoint and a planned accrual of 700 patients. Results from these trials will help to further delineate the feasibility and technical issues mentioned above. The results of the current study demonstrated the feasibility of our biomarker-based decision algorithm in a multiinstitutional cooperative group environment for patients with surgically resected NSCLC. We identified that the current practice of evaluation and treatment for these patients may present an obstacle to rapid molecular-based decision-making. Although encouraging efficacy data emerged from this trial bioassays that specifically measure platinum-induced DNA damage repair must be developed before further clinical trials are launched that seek to tailor the use of these agents. FUNDING SUPPORT Supported by National Cancer Institute grants CA014028 CA016385 CA020319 CA022453 CA027057 CA032102 CA 035090 CA035119 CA035178 CA035261 CA035431 CA 038926 CA042777 CA045377 CA045560 CA045807 CA0 46113 CA046368 CA046441 CA063844 CA063848 CA0 67575 CA067663 CA073590 CA074647 CA076429 CA10 5409 and CA129343. CONFLICT OF INTEREST DISCLOSURES Dr. Bepler has a patent pending for the use of RRM1 and ERCC1 as biomarkers of treatment benefit for therapeutic decision-making in patients with cancer. REFERENCES 1 Arriagada R Bergman B Dunant A Le Chevalier T Pignon JP Vansteenkiste J International Adjuvant Lung Cancer Trial Collaborative Group Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer N Engl J Med 2004 350 351 360 14736927 2 Winton TL Livingston R Johnson D Vinorelbine plus cisplatin versus observation in resected non-small-cell lung cancer N Engl J Med 2005 352 2589 2597 15972865 3 Strauss GM Herndon J Maddaus MA Randomized clinical trial of adjuvant chemotherapy with paclitaxel and carboplatin following resection in stage IB non-small-cell lung cancer: report of the Cancer and Leukemia Group B protocol 9633 [abstract] Proc Am Soc Clin Oncol 2004 23 Page Abstract 7019 4 Strauss GM Herndon JE Maddaus MA Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small-cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B Radiation Therapy Oncology Group and North Central Cancer Treatment Group Study Groups J Clin Oncol 2008 26 5043 5051 18809614 5 Dabholkar M Vionnet J Bostick-Bruton F Yu JJ Reed E Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy J Clin Invest 1994 94 703 708 8040325 6 Metzger R Leichman CG Danenberg KD ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy J Clin Oncol 1998 16 309 316 9440758 7 Lord RV Brabender J Gandara D Low ERCC1 expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer Clin Cancer Res 2002 8 2286 2291 12114432 8 Olaussen KA Dunant A Fouret P DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy N Engl J Med 2006 355 983 991 16957145 9 Reynolds C Obasaju C Schell MJ Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer J Clin Oncol 2009 27 5808 5815 19884554 10 Davidson JD Ma L Flagella M Geeganage S Gelbert LM Slapak CA An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines Cancer Res 2004 64 3761 3766 15172981 11 Bergman A Eijk P van Haperen V In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as a major determinant Cancer Res 2005 65 9510 9516 16230416 12 Bepler G Kusmartseva I Sharma S RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer J Clin Oncol 2006 24 4731 4737 16966686 13 Ceppi P Volante M Novello S ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine Ann Oncol 2006 17 1818 1825 16980606 14 Bepler G Sharma S Cantor A RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer J Clin Oncol 2004 22 1878 1885 15143080 15 Simon GR Sharma S Cantor A Smith P Bepler G ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer Chest 2005 127 978 983 15764785 16 Zheng Z Chen T Li X Haura E Sharma A Bepler G The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer N Engl J Med 2007 356 800 808 17314339 17 Camp RL Chung GG Rimm DL Automated subcellular localization and quantification of protein expression in tissue microarrays Nat Med 2002 8 1323 1327 12389040 18 Bepler G Williams C Schell MJ Randomized international phase III trial of ERCC1 and RRM1 expression-based chemotherapy"

"Age is associated with the prognosis of glioma patients but there is no uniform standard of agegroup classification to evaluate the prognosis of glioma patients In this study we aimed to establish an age groupclassification for risk stratification in glioma patientsMethods patients diagnosed with gliomas at Nanfang Hospital between and were enrolled TheWHO grade of glioma was used as a dependent variable to evaluate the effect of age on risk stratification Theevaluation model was established by logistic regression and the Akaike information criterion AIC value of themodel was used to determine the optimal cutoff points for ageclassification The differences in gender WHOgrade pathological subtype tumor cell differentiation tumor size tumor location and molecular markers betweendifferent age groups were analyzed The molecular markers included GFAP EMA MGMT P53 NeuN Oligo2 EGFRVEGF IDH1 Ki67 PR CD3 H3K27M TS and 1p19q statusResults The proportion of men with glioma was higher than that of women with glioma vs Analysisof age showed that appropriate classifications of age group were “ years old pediatric group “ years oldyouth group “ years old middleaged group and ‰¥ years old elderly groupThe proportions ofglioblastoma and large tumor size “ cm increased with age p p respectively Analysis of thepathological molecular markers across the four age groups showed that the proportion of patients with larger than area of Ki67 expression or positive PR expression increased with age p p respectivelyConclusions Appropriate classifications of the age group for risk stratification are “ years old pediatric group“ years old young group “ years old middle age group and ‰¥ years old elderly group This agegroup classification is effective in evaluating the risk of glioblastoma in glioma patientsKeywords Glioma Age group classification Risk stratification Personalized treatment Correspondence hgl1020163com Zhiying Lin and Runwei Yang contributed equally to this work1Department of Neurosurgery Nanfang Hospital Southern MedicalUniversity No Guangzhou Avenue North Guangzhou Guangdong China2The Laboratory for Precision Neurosurgery Nanfang Hospital SouthernMedical University Guangzhou Guangdong ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLin BMC Neurology Page of BackgroundOver the past years the incidence of primary malignant brain tumors has increased at an annual rate of “ with an especially higher rate in the elderly population [] Glioma accounts for approximately of allcentral nervous system CNS tumors and of malignant primary brain tumors [] According to the World Health anization WHO classification of tumors ofthe CNS gliomas were classified into fourgrades WHO grade I to IV based on histologic criteria[] WHO grades I and II gliomas are recognized as lowgrade gliomas LGG and grades III and IV are considered highgrade gliomas HGG [] In particular glioblastoma GBM WHO grade IV is the most commonmalignant tumor of the CNS accounting for ofprimary malignant the CNS tumors and of all gliomas [] The median survival of GBM patients is approximately months even after receiving multimodaltherapies that include maximal surgical resection withthe preservation of neurological functions followed byadjuvant radiotherapy and chemotherapy []Gliomas can occur at any age with various incidencesat different ages as reported in populationbased studies[ ] LGG is the most common brain tumor in children while HGG is the most frequent brain tumor inadults [] Tumors in the supratentorial areas of thebrain cerebral hemispheres and midline structuresabove the tentorium were most frequent in adults whilesubtentorial brainstem and cerebellum tumors weremore common in young children than in adolescentsand adults [] Besides increasing studies have assessedage a prognostic factor There are differences in prognosis among patients of different ages even with the samediagnosis A singlecenter review of patients withintracranial anaplastic oligodendroglioma showed thatthe median survival time of patients younger than years old was significantly longer than that of patientsolder than years old [] Other studies have shownthat age was an important prognostic factor in additionto KPS score surgical scope and histology [ ]Therefore for patients diagnosed with glioma by imaging examination and auxiliary examination it is necessary to consider the age of the patients to performpersonalized treatment for better outcomesHowever there is no uniform age criterion for groupingglioma patients for personalized treatment [] Some glioma patient cohorts were divided into different age groupsaccording to fixed age intervals [] some were dividedinto two groups based on a certain age point [] andothers were divided based on the overall survival OS ofthe patients [] Different criteria for age grouping haveled to inconsistent s regarding the prognosticvalue of age Some studies showed that age was not aprognostic factor in patients with glioma [ ] whileanother populationbased glioblastoma study with five agegroups years “ years “ years “ yearsand years showed that the OS of young patients years was significantly longer than that for elderly patients years median months vs months p [] Agerelated studies involving a large numberof glioma patients have yielded some relevant results [] but the age grouping criteria for these studies are influenced by several clinical factors such as the tendencyof clinical researchers Therefore there is an urgent needto establish a more appropriate age group classificationcriterion for better management of glioma patientsFor this purpose we conducted a retrospective studycollecting clinical data from patients with histologically proven gliomas in Nanfang Hospital between and Based on this cohort we established a methodof age group classification according to WHO grade forrisk stratification in glioma patients and investigated thecharacteristics of different age groups in terms of genderWHO grade pathological subtype tumor cell differentiation tumor size tumor location and pathological molecular markersMethodsData collectionA total of patients diagnosed with gliomas bypathological examination after surgery from to in Nanfang Hospital were enrolled in this studyThe clinical data for age gender pathological diagnosisaccording to the WHO Central nervous systemtumor Classification anatomic location of gliomatumor size and pathological molecular markers werecollected Supplementary Table S1The terminology of the anatomic location of gliomaused in this study was based on the Central BrainTumor Registry of the United States CBTRUS Brainand other Central Nervous System Tumor Site Groupings We recognize that with the WHO classification of central nervous system tumors many of thehistological diagnostic criteria have undergone majorchanges and steps have been taken to align their histological grouping scheme with the WHO standardsThe pathological diagnosis included histological classification WHO grade and molecular expression Thepathological molecular markers included GFAP EMAMGMT wtP53 NeuN Oligo2 EGFR VEGF IDH1 Ki and ATRXfluorescence in situhybridization FISH detection of 1p19q was also included All pathological information was collected fromthe hospital medical records systemIn additionCalculation of age group cutoff pointsDummy variables were established by age groups of 1Iyears old and I82 years old I any age between and 0cLin BMC Neurology Page of The established dummy variables were consideredas independent variables and a logistic regression modelwas established according to whether the patients werehighgrade glioma or WHO IV grade glioma whichwere set as dependent variables The AIC was calculatedto determine the best cutoff point for age among allmodels The model with the lowest AIC value wasregarded as the best model The results showed that thediagnostic age classification criterion was “ years oldand ‰¥ years old The probability of highgrade gliomaor WHO IV grade glioma in the age group ‰¥ years oldwas greater than that in the age group “ years old vs vs respectively Supplementary Table S2tolerance and treatmentOwing to the differences in the epidemiology betweenadults and pediatric glioma patients the differences insurgicalregimens betweenmiddleaged and elderly patients and the various prognoses of patients of different groups even with the samediagnosis only two age groups for the classification ofglioma patients were not sufficient in clinical practiceTherefore these two groups were subdivided into fourgroups First dummy variables were created by agegroups of 0I years old and I47 years old I any age between and The established dummy variables wereconsidered as independent variables and a logistic regression model was set up according to whether gliomapatients were high grade glioma or WHO IV grade glioma The AIC value for each model was calculated Themodel with the smallest AIC value was regarded as thebest model According to whether the patient sufferedfrom WHO IV glioma the diagnostic age classificationcriteria were “ years old pediatric group and “years old young group According to whether the patient was suffered from highgrade glioma the diagnosticage classification criteria were “ years old pediatricgroup and “ years old young group The evidencesuggeststhe difference between the biologicalspectrum of the disease may be reflected in the diagnostic age with the majority of the pediatric group belonging to the category described by Paugh et al[]Although some of the molecular abnormalities encountered in HGG in children are reminiscent of secondaryglioblastomas these tumors rarely originate from existing LGGs [] Finally years old was chosen as theage for distinguishing the pediatric group from the adultgroupthatSecond dummy variables as independent variableswere established by age groups of 48I years old and ‰¥ Iyears old I any age between and The cutoff ofthe model with the minimum AIC value was calculatedby the same method described above The resultingdiagnostic age classification criterion was “ yearsold middleaged group and ‰¥ years old elderlygroup The probability of highgrade glioma or WHOIV grade glioma in the age group ‰¥ years was greaterthan that of the age group “ years oldStatistical analysisThe SPSS statistical software package version IBMCorp was used for all analyses The statistical significance level was set as p Note that reported percentages may not add up to due to roundingCategorical variables are shown as numbers and percentages while continuous variables are shown as the meanand standard deviation SD Pearson™s chisquare testwas performed to compare the categorical dataResultsAnalysis of demographic and clinical characteristicsThe study population comprised male patients and female patients The ratio of malesto females was The age range was to years oldand the mean age was years old SD years oldThere were patients were classified as WHOgrade I patients were classified as WHOgrade II patients were classified as WHOgrade III and patients were classified asWHO grade IV Supplementary Figure S2According to the WHO classification of tumorsof the CNS the glioma patients diagnosed andtreated at Nanfang Hospital were subdivided into histologically distinct types of primary glioma Astrocytomas accounted for approximately n of allgliomas The average diameter of glioma was cmSD cm Gliomas mostly occurred in the frontallobe n and temporal lobe n GBM represented the majority of gliomas n The distribution of tumor sites showed that cases occurred in the brain casesoccurred in the spinal cord and cauda equina and cases involved the spinal cord cauda equina andbrain Detailed information for this cohort of glioma patients is recorded in Supplementary Table S1The median age at diagnosis for all primary glioma tumors was years old As shown by the cumulativecurves of the proportion of gliomas across four WHOgrades gliomas of higher grades tended to be diagnosedat older ages Fig 1a p The average age at diagnosis of WHO grade IV glioma was while WHOgrade I gliomas were diagnosed at years with anage gap of more than years Fig 1b The average agesat diagnosis of WHO grade II and III were and years respectively Fig 1b In addition we compared the average age at diagnosis of various pathological subtypes of glioma We found that anaplasticastrocytoma WHO grade III was diagnosed at an olderage than that ofindividuals diagnosed with diffuse 0cLin BMC Neurology Page of Fig Cumulative age distribution and T test of the average age at diagnosis of different types of glioma a Cumulative age distribution of WHOIIV grade glioma the mean age of glioma patients increases with the WHO grade WHO I years WHO II years WHO III years andWHO IV years respectively b The diagnosed age boxplot figure of WHO IIV grade glioma c Cumulative age distribution of anaplasticastrocytoma and diffuse astrocytoma there is likely for an earlier manifestation in diffuse astrocytoma d The average age at diagnosis ofanaplastic astrocytoma and diffuse astrocytoma e Cumulative age distribution of Oligodendroglioma and anaplastic oligodendroglioma most ofoligodendroglioma and anaplastic oligodendroglioma arise in adults with peak incidence in patients aged “ years f The diagnosed ageboxplot figure of oligodendroglioma and anaplastic oligodendroglioma g Cumulative age distribution of Oligoastrocytoma and anaplasticoligoastrocytoma the median ages of patients with oligoastrocytoma are years The median age of patients with anaplastic oligoastrocytomais years h The diagnosed age boxplot figure of oligoastrocytoma and anaplastic oligoastrocytomaastrocytoma WHO grade II Fig 1c and d vs years respectively p With a similar trendanaplastic oligodendroglioma WHO grade III was diagnosed at a median age of years and oligodendroglioma WHO grade II was diagnosed at a median age of years Fig 1e and f p Besides oligoastrocytoma WHO grade II and anaplastic oligoastrocytomaWHO grade III were diagnosed at average ages of and years respectively Fig 1g and h p Isocitrate dehydrogenase IDH1 is a vital marker for themolecular classification of glioma In this cohort whenanalyzing the average age at diagnosis of different IDH1phenotypes by using the whole cohort no significant differences were observed Supplementary Figure S1B andD however IDHwt GBM was diagnosed at an olderage than that of individuals diagnosed with IDHmutGBM Supplementary Figure S1A and C vs respectively p These results indicated that theage at diagnosis was closely correlated with the WHOgrade and pathological subtypes of gliomaEstablishment of age group classification cutoffAge and positive area of Ki67 and wtP53 showed greatvalue for the diagnosis of WHO grade IV glioma andhighgrade glioma Fig 2a and b The status of Ki67and P53 could be assessed only after surgery of biopsywhile the information of age could be obtained beforesurgery Therefore age could be an earlier factor for theevaluation of patients in clinical practice We thensought to establish an age group classification for bettermanagement of patients according to the AIC methodmentioned in the section of œmethod Glioma patientswere divided into four age groups “ years oldpediatric group “ years old youth group “years old middleaged group and ‰¥ years old elderlygroup of patients were “ years old pediatricgroup were “ years old middleaged group were “ years old youth group and were ‰¥ years old elderly group The proportion ofprimary WHO grade IV gliomas and larger tumor sizeslarger than cm increased with age Fig 2c and ghowever the proportions of glioma of astrocyte differentiation only include WHO grade IIII and ependymalcells differentiation decreased with age Fig 2d and fMost of the gliomas of oligodendrocyte differentiationwere found in “ age group Fig 2eTo examine the value of this age group classificationin risk stratification of GBM we collected data from patients in the Chinese Glioma Genome Atlas CGGAdatabase and calculate the proportion of different gliomagrade in four age groups respectively The sensitivity ofpredicting WHO grade IV was the specificity was 0cLin BMC Neurology Page of Fig ROC curve of the sensitivity and specificity for diagnosing WHO IV glioma a and high grade glioma b Age ki67 and positive area ofwtp53 have great value for the diagnosis of WHO grade IV glioma and highgrade glioma The proportion of WHO grade IV glioma c astrocytedifferentiation d oligodendrocyte differentiation e ependymal cells differentiation f and cm of tumor size g in four age groupsAccording to the discriminant classification of whether the pathological diagnosis of the patients was WHO grade IV or not the predictionprobability was taken as the discriminant dividing point and the total judgment rate was h and the total judgment rate was p Fig 2hAnalysis of the pathological subtypes of glioma acrossfour age groupsIn the pediatric group the proportion of pilocytic astrocytoma was while GBM accounted for the largestproportion in the youth group middleage group andelderly group and respectively Fig and Supplementary Figure S3 Pilocytic astrocytomapleomorphic xanthoastrocytoma ependymoma anaplastic ependymoma choroid plexus papilloma atypicalchoroid plexus papilloma and ganglioglioma are predisposed to patients in pediatric group Diffuse astrocytoma diffuse midline glioma H3K27Mmutant gliomaoligodendroglioma oligoastrocytoma and myxopapillaryependymoma commonly occurred in youth group Anaplastic astrocytoma anaplastic oligodendroglioma andanaplastic oligoastrocytoma were more likely to occur inmiddleage group GBM and anaplastic gangliogliomawere more likely to occur in elderly group p0001The proportions of anaplastic oligodendroglioma andanaplastic ganglioglioma increased with age Ependymoma gradually decreased in the younger age groupsFig Analysis of glioma cell differentiation size and anatomiclocation across four age groupsPatients aged ‰¥ years old were predisposed to gliomasof astrocyte differentiation Patients aged “ yearsold were predisposed to gliomas of oligodendrocyte andhybrid cell differentiation Patients aged “ years oldwere predisposed to gliomas of ependymal cell and othercells differentiation Supplementary Table S2 p The proportion of tumors with sizes of “ cm decreased with age however the proportion of tumorswith sizes ranging from to cm was larger in oldergroups Supplementary Table S2 p In the pediatric group the common locations of gliomas were the cerebellum and ventricle accountingfor and respectively Supplementary Tablein the youth and middleage groupsS3 Howeverlobe accounted for the largest proportionthe frontalSupplementary Table S3 p In the elderlygroup the proportion of tumors in the frontallobeand temporal lobe was higher than that in the otherlocations Supplementary Table S3 and respectivelyAnalysis of molecular marker expression in four agegroupsThe proportion of positive expression of glial fibrillaryacidic protein GFAP was more than in all agegroups Detailed information is recorded in Supplementary Table S2 The proportion of positive expression ofIDH1wt Ki67 and Oligodendrocyte transcription factor Oligo2 increased with age The proportion ofpositive expression of epithelial membrane antigenEMA vascular endothelial growth factor VEGF andMGMTO6methylguanineDNA methyltransferasewere maximalthein the pediatric group while 0cLin BMC Neurology Page of Fig Histological distribution by Age groups a Histological distribution by “ years old group b Histological distribution by “ years oldgroup c Histological distribution by “ years old group and d Histological distribution by ‰¥ years old group In the “ age group Theproportion of pilocytic astrocytoma in the histological distribution was however glioblastoma accounted for the largest proportion of theage group “ years old “ years old and ‰¥ years old with and respectivelyFig Composition changes of pathological subtypes across four age groups 0cLin BMC Neurology Page of proportion of positive expression of neuronal nucleiNeuN and epidermal growth factor receptor EGFRwere highest in the middleage group Fig Besideswe analyzed the expression of gliomaassociated genesin homogeneous groups including subgroups of different cell origins and different molecular subtypes suchas EGFRpositive and EGFRnegative gliomas The results revealed great heterogeneity across the four agegroups Supplementary Figure S4 S5 S6 S7 S8 S9S10 S11 Supplementary Table S4S5DiscussionClinical and biological data clearly indicate thatthecharacteristics and outcomes of malignant gliomas differsignificantly between adults and children [] A numberof studies have showed that the tumorprone locationshistopathology prognosis and some molecular markersare different in glioma patients of different ages [ ]Growing research has shown that the molecular characteristics of GBM in elderly patients are more aggressivethan those in young patients [] Childhood GBMFig The glioma heatmap of 10gene signatures by gene expression subtype Representative genes are shown for each subtype a Heatmap ofpediatric group b Heatmap of youth group c Heatmap of middleage group d Heatmap of elderly group 0cLin BMC Neurology Page of displayed on average considerably fewer DNA copynumber changes than histologically similar adult tumor[“] In addition the prognosis of glioma is particularly severe in older adults [ ] The clinical practicepatterns show that with increasing age the applicationof surgical resection radiotherapy and chemotherapy decreases [“] Nevertheless some elderly patients withglioblastoma can benefitfrom these therapies []These elderly patients will receive aggressive treatmentwith radiation or chemotherapy When consideringtreatment options for children with gliomas neurosurgeons will try to avoid the deleterious effects of radiotherapy on the developing brains of children Minimaldysfunction resulting from glioma and treatment shouldbe achieved as much as possible with the expectation ofchildren living to adulthood [] Moreover age isregarded as an important factor related to the prognosisof glioma patients Thereforefor patients diagnosedwith glioma age should be taken into consideration toperform personalized treatment for a better outcomeHowever the criterion for appropriately dividing agegroups of glioma patients remains an unresolved clinicalproblemA large number of studies used different age groupings and these studies led us to differential sabout the prognosis value of age in glioma patients [ ] These contradictory s could be partlyexplained by the difference in age classification criteriabetween different studies In one study a multivariateCox regression model with different cutoff points wasused to analyze the effect of age on OS but only threeage groups were compared and univariate analysis wasperformed using prognostic factors as a classification criterion [] OS is a good indicator for evaluating patientoutcomes but confounding factors such as tumor sizetumor location surgical resection extent and patientcompliance might impair the accuracy of the relationship between age and OSTo avoid the disturbance of confounding factors asmuch as possible our study used the WHO grade of glioma as a dependent variable to assess the prognosis ofglioma patients The classification criteria for glioma patients based on age were “ years old pediatric groupand “ years old youth group “ years oldmiddleaged group and ‰¥ years old elderly groupThis age group classification can be used for preliminaryevaluation of newlydiagnosed glioma patients and helpsto perform precise management in clinical practice according to age group Besides we found that EGFRpositive expression was more common in the middleage group and the EGFR expression in IDH1mut gliomas was more apparent Therefore patients withIDH1mut glioma aged “ years old might benefitfrom EGFR inhibitor therapy Based on this age groupclassification we further analyzed the characteristics ofWHO grade tumor size tumor histology and anatomical location among the four age groups We found thatthe proportion of WHO grade IV gliomas and positiveexpression of Ki67 Oligo2 and IDHwt increased significantly in elderly age groups In addition in the olderage group more patients suffered from a heavy tumorburden tumor size cm Regarding the histology ofglioma pilocytic astrocytoma is the most common inchildren while glioblastoma accounts for the largest proportion of adult groups Many studies have demonstrated that patients with a higher grade of glioma havea worse outcome [] Moreover a larger tumor burdenmight cause a higher risk of functional deficits includingmotor dysfunction impaired communication ability ordecline in neurocognitive function [] Therefore theprognosis of patients with gliomas can initially be evaluated according to age On the other hand patientsgrouping according to age has been widely used in clinical studies but there is no uniform standard of agegroup classification for patients with glioma The agegroup established on the basis of objective pathologicaldiagnosis in this study will be helpful for clinical trialsdesign in the futureGlioma especially glioblastoma is a highly heterogeneous malignancy In addition to the marked heterogeneity of tumor size and histopathology the heterogeneityof the molecular characteristics of tumors is becomingincreasingly important and is reported in several studiesAccording to the WHO classification glioma isfirst classified according to histologicalfeatures andthen more subtypes are classified according to molecularcharacteristics There are a variety of indicators that arewidely used in clinical practice such as GFAP EMAMGMT P53 NeuN Oligo2 EGFR VEGF IDH1 Ki671p19q and these indicators are highly correlated withthe prognosis of the patients [“] Agedependentoccurrence and the effects of different biological markershave been reported in malignancies [] For examplethe association between age and tumor grade Ki67markers apoptosis index EGFR expression and erbB2expression has been reported in breast cancer [] Astudy indicated that the prognostic effects of P53 1pand CDKN2Ap16 alterations are dependent on patientage [] Increasing translational studies have significantly advanced the understanding of glioma pathogenesis and have identified several prognostic factorsHigher tumor grade older age [] and increased expression of molecular biomarkers such as P53 []MGMT [] PR [] IDH1wildtype [] H3K27Mmutation of pediatric HGG [ ] and Ki67 []were related to poorer prognoses Analysis of the pathological molecular markers acrossfour age groupsshowed that the proportion of patients with larger than 0cLin BMC Neurology Page of area of Ki67 positive expression or PR positive expression increased with age Other molecular markersGFAP EMA NeuN EGFR IDH1 CD3 and H3K27Mshowed great heterogeneity among the four age groupsGender age anatomic location of the tumor size oftumor and molecular markers are simple and objectiveparameters that can be collected easily in clinical practice or clinical studies on patients with glioma Our research can provide clinicians with a simple method toevaluate the prognosis of glioma patients and help topromote the personalized management of glioma patients In addition for some clinical trials that need todivide participants of glioma into different groups thisage group classification based on WHO grade will bemore objective However this study was limited by thesample size and these data were retrospective Hospitalbased retrospective studies may lead to certain selectionbiases Another limitation of this study was that we didnot include patients with postoperative recurrence Further validation of our results will require multicenterprospective studies with larger sample sizesConclusionOur research indicated that the classification criteriabased on the age for glioma patients were “ years oldpediatric group “ years old youth group “years old middleaged group and ‰¥ years old elderlygroup Our cohort indicates that pilocytic astrocytomaaccounts for the largest proportion in the “ year agegroup while GBM accounts for the largest proportion inthe other three age groups Besides the proportion oftumors of “ cm in size or with Ki67 increaseswith WHO grade This age group classification will helpto improve the diagnosis personalized treatment andclinical trial design involved patients with gliomaSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1288302001888wAdditional file Figure S1 Cumulative age distribution and T test ofthe average age at diagnosis of glioma A Cumulative age distribution ofIDH1wt glioma and IDH1mut glioma B Cumulative age distribution ofIDH1wt glioma and IDH1mut glioma C The diagnosed age boxplot figure of IDH1wt GBM and IDH1mut GBM D The diagnosed age boxplotfigure of IDH1wt GBM and IDH1mut GBMAdditional file Figure S2 Constituent ratios of four age groups Theproportion of patients in the four age groups “ “ “ and ‰¥ years oldAdditional file Figure S3 Distribution by Age groups of otherhistology A “ years old B “ years old C “ years old D‰¥ years old subependymal giant cell astrocytoma subependymomaangiocentric glioma chordoid glioma of the third ventricle anaplasticganglioglioma desmoplastic infantile astrocytoma and gangliogliomawere not analyzed because the total number of patients was no morethan threeAdditional file Figure S4 Heatmap of 10gene signatures by geneexpression subtype Representative genes are shown for each subtype AHeatmap of all glioma B Heatmap of all GBM C Heatmap of pediatricgroup D Heatmap of youth old group E Heatmap of middleage groupF Heatmap of elderly groupAdditional file Figure S5 The heatmap of glioma derived fromastrocyte differentiation only including WHO grade I III A Heatmap ofpediatric group B Heatmap of youth group C Heatmap of middleagegroup D Heatmap of elderly groupAdditional file Figure S6 The heatmap of glioma derived fromoligodendrocyte differentiation A Heatmap of pediatric group BHeatmap of youth group C Heatmap of middleage group D Heatmapof elderly groupAdditional file Figure S7 The heatmap of glioma derived fromependymal cells differentiation A Heatmap of pediatric group BHeatmap of youth group C Heatmap of middleage group D Heatmapof elderly groupAdditional file Figure S8 The heatmap of glioma with EGFRpositive expression A Heatmap of pediatric group B Heatmap of youthgroup C Heatmap of middleage group D Heatmap of elderly groupAdditional file Figure S9 The heatmap of glioma with EGFRnegative expression A Heatmap of pediatric group B Heatmap ofyouth group C Heatmap of middleage group D Heatmap of elderlygroupAdditional file Figure S10 The heatmap of IDH1mut glioma AHeatmap of pediatric group B Heatmap of youth group C Heatmap ofmiddleage groupAdditional file Figure S11 The heatmap of IDH1wt glioma AHeatmap of pediatric group B Heatmap of youth group C Heatmap ofmiddleage groupAdditional file Table S1 Clinical and molecular characteristics ofpatients with gliomas n Additional

"the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen therefore is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance. Hsp70 apoptosis ibuprofen The human Hsp70 family includes ?8 highly homologous members that differ from each other by their intracellular localization and expression patterns.1 Among them the major stress-inducible Hsp70 (also called Hsp72) has an essential role in cell survival under stressful conditions. Compared with its normal counterpart Hsp70 is often overexpressed in various cancer cells and is suspected to contribute to the development of tumours.2 3 Indeed the expression of Hsp70 in certain cancer types has been correlated with poor prognosis and resistance to chemotherapy.45 6 Tumour cells often express several proteins that when abnormally elevated render the tumour resistant to apoptosis.7 Previous studies have confirmed not only that Hsp70 is cytoprotective but also that it interferes effectively with cell death induced by a wide variety of stimuli including several cancer-related stresses. Hsp70 is a potent inhibitor of the stress-activated kinase pathway and apparently blocks apoptotic signals via interactions with JNK Ask1 and SEK1.8910 11 Hsp70 is also a negative regulator of the mitochondrial pathway of apoptosis. Much of the focus on the antiapoptotic function of Hsp70 has been on events that occur after the disruption of the mitochondria. Hsp70 prevents the recruitment of procaspase-9 to the apoptosome and its functional complex formation by direct interaction with apoptotic protease-activating factor 1 (Apaf-1).12 13 Furthermore Hsp70 inhibits the activation of caspase-3 and the cleavage of caspase-3 targets such as ICAD and GATA-1.14 15 On the other hand recent studies have reported that Hsp70 can prevent apoptosis upstream of the mitochondria by inhibiting events which ultimately permeabilize the mitochondrial outer membrane such as the activation of Bax.16 17 As a result of the inhibition by Hsp70 of the apoptosis induced by several anticancer drugs as well as by other stimuli we hypothesized that cancer cells would be sensitized to the induction of apoptosis by the neutralization of Hsp70. Hsp70 has been indeed targeted with pharmaceuticals such as triptolide quercetin and KNK437 which downregulate its expression.1819 20 Although they have prevented the progression of various cancer cells in vitro and in vivo21 22 the optimal clinical use of these small Hsp70 inhibitors singly or combined with other chemotherapeutics remains a challenge. Our overall objective was to pharmacologically control the levels of Hsp70 and increase the effectiveness of anticancer drugs. Several experimental and epidemiologic studies and clinical trials have observed a powerful chemopreventive activity exerted by nonsteroidal anti-inflammatory drugs (NSAIDs).23 24 The anti-carcinogenic properties of NSAID have been attributed to their inhibition of cyclooxygenase (COX) enzymes. However much higher doses of NSAID are needed to obtain an antitumoural effect than to inhibit COX25 suggesting that they also act via COX-independent mechanisms. On the other hand NSAIDs such as aspirin salicylate and sulindac sulphide inhibit the proliferation of cells and induce apoptosis in various cancer cell lines which is considered an important component of their antitumoural activity and increased sensitization of cancer cells to anticancer drugs.262728 29 There is currently interest in the ability of NSAID to directly lower the levels of antiapoptotic molecules such as the Bcl-2 family30 and 14-3-3 protein31 which inhibits the intrinsic mitochondria-dependent apoptosis in various cancer cells. Therefore the NSAID-induced dysfunction of antiapoptotic proteins prompted us to examine whether other antiapoptotic molecules including Hsp70 might also be targets in the prevention of tumour progression by NSAID. In this study we show that ibuprofen is a potent inhibitor of Hsp70 which significantly suppresses its expression by depleting heat shock factor 1 (HSF1) in lung adenocarcinoma-derived A549 cells. The downregulation of Hsp70 by ibuprofen sensitized the cells to cisplatin which was associated with the enhancement of cisplatin-induced apoptotic signalling. Ibuprofen did not only facilitate postmitochondrial events including the activation of cisplatin-induced caspase-9 but also the activation of Bax causing the release of cytochrome c. Besides the demonstration of a similar increase in the sensitivity of A549 cells to cisplatin conferred by Hsp70 knockdown and ibuprofen these observations indicate that ibuprofen accelerates cisplatin-mediated apoptosis at multiple steps of the mitochondrial apoptotic pathway via the inhibition of Hsp70. We conclude that ibuprofen is a potential chemotherapeutic agent which might enable (a) the use of lower less toxic does of cisplatin and (b) the design of a new combination treatment of lung cancer. Results Ibuprofen suppresses the expression of Hsp70 in lung adenocarcinoma cells To define the role of Hsp70 in promoting the formation of tumours we first examined its expression in human lung cancer cell lines. Compared with BEAS-2B a human non-malignant bronchial epithelial cell line the expression levels of Hsp70 in lung cancer cells such as A549 and H358 adenocarcinoma were notably higher (a). As in previous studies which showed an increased expression of Hsp70 in various types of human cancers including breast pancreas and colon we found that Hsp70 is also dysregulated in lung cancer cells. In this study we screened conventional NSAID in search of a new pharmacologic inhibitor which neutralizes Hsp70 as they induce apoptosis in cancer cells by selectively downregulating antiapoptotic proteins. The expression of Hsp70 after the exposure of A549 cells to various NSAID in non-toxic concentrations was analyzed by immunoblot. Ibuprofen in a 400-?M concentration decreased the expression of Hsp70 by 23% in comparison with untreated cells whereas other NSAID had no effect (). b shows the decrease in Hsp70 protein and mRNA levels in A549 cells after treatment with various concentrations of ibuprofen versus no apparent decreases in Hsc70 and Actin. Ibuprofen also decreased the expression of Hsp70 in H358 a human lung adenocarcinoma cell line in a dose-dependent manner (c). These results suggest that ibuprofen decreases the expression of Hsp70 in various lung cancer cell lines. Ibuprofen enhances the apoptosis induced by cisplatin by suppressing Hsp70 As ibuprofen prominently inhibited the expression of Hsp70 we next examined its effect on the proliferation of cancer cells. We observed no significant change in the viability of A549 and H358 cells after the exposure to ?800??M concentrations of ibuprofen alone which downregulates Hsp70 (a) while the exposure to 1.0?mM concentration of ibuprofen caused cell death. Combined these observations indicate that the downregulation of stress-inducible Hsp70 was insufficient to cause the death of A549 and H358 cells. There is evidence that the inhibition of anti-apoptotic molecules such as Hsp70 increases the sensitivity of tumour cells to anticancer drugs thus improving the outcomes of chemotherapy. To study the therapeutic potential of ibuprofen we examined whether its antitumoural effects are synergistic with those of cisplatin widely used in the treatment of lung adenocarcinoma. When we measured the survival of A549 (top of b) and H358 (bottom of b) cells exposed to increasing concentrations of cisplatin incubated in presence versus absence of ibuprofen the latter prominently magnified the apoptosis induced by cisplatin a synergistic effect confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL) staining (c). To ascertain the effects conferred by the expression of Hsp70 on cell death while excluding all effects of ibuprofen unrelated to Hsp70 we weakened the expression of Hsp70 by RNA interference (RNAi) (d) and measured its effects on the apoptosis induced by cisplatin. The inhibition of Hsp70 decreased the viability of cisplatin-treated cells by approximately 20% (e). Transfections with scrambled siRNA serving as a control showed no increase in cell death mediated by cisplatin. Cisplatin had no effect on the expression of Hsp70 (g). We quantified the number of apoptotic cells in ibuprofen- and/or cisplatin-treated cultures using the CF488A-annexin V methods. Although cisplatin alone induced apoptosis in 10.2% of A549 cells the co-treatment with ibuprofen increased the percentage of apoptotic cells to 34.0% (f). These observations suggest that ibuprofen sensitizes A549 cells to cisplatin by decreasing the expression of Hsp70. Ibuprofen decreases the expression of Hsp70 via transcriptional inactivation The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis described earlier revealed a decrease in RNA level following treatment with ibuprofen suggesting that the expression of Hsp70 can be downregulated at the transcriptional level. After the recently discovered inhibition by its antagonists of the transcription of Hsp70 in cancer cells by blockade of the activation of HSF118 20 (which is often upregulated and constitutively activated in tumour formation) we studied the effects of ibuprofen on HSF1 in A549 cells. We first performed a ChIP assay to explore whether the inhibitory effect of ibuprofen is at the level of HSF1 DNA binding. As expected we found an unequivocal association between HSF1 and the Hsp70 gene promoter containing the HSE site in ibuprofen-untreated cells (Figure 3a). It is noteworthy that ibuprofen eliminated this binding (Figure 3a) suggesting that it inhibits the expression of Hsp70 via the action of HSF1. This also suggests that ibuprofen blocks the binding of HSF1 chromatin or the steps which precede in several processes needed to activate HSF1. Therefore we broadened our analysis to examine the effect of ibuprofen on the expression of HSF1. Compared with unexposed control cells the HSF1 mRNA level was significantly lower in cells exposed to ibuprofen (bottom of Figure 3b). Consistent with its effect on the expression of mRNA ibuprofen also decreased the expression of HSF1 protein in a dose-dependent fashion (top of Figure 3b). To confirm the inhibition of HSF1-mediated Hsp70 by ibuprofen we lowered the amounts of HSF1 present in A549 cells by RNAi and studied its effect on the expression of Hsp70. The treatment of cells with HSF1 dsRNA decreased the Hsp70 level compared with that measured in cells untreated with dsRNA (Figure 3c). Ibuprofen decreased the expression of HSF1 by 16% in comparison with untreated cells whereas other NSAID had no effect (Table 2). Overall these observations indicate that ibuprofen inhibited the expression of Hsp70 by depleting the HSF1 in A549 cells. Ibuprofen accelerates the mitochondrial apoptotic process induced by cisplatin Several studies have found that mitochondria might be a direct and important target of cisplatin in sensitive cells.32 33 We studied the effects of ibuprofen on the depolarization of mitochondrial membranes and the cytochrome c release induced by cisplatin. A549 cells with or without cisplatin were incubated in absence or presence of ibuprofen and stained with JC-1. Treatment with cisplatin and ibuprofen lowered the mitochondrial membrane potential manifest by an attenuated red and an enhanced green mitochondrial fluorescence (Figure 4a lower right panel) compared with that observed with cisplatin alone (Figure 4a upper right panel) while control (Figure 4a upper left panel) or ibuprofen alone (Figure 4a lower left panel) produced the red-dotted staining pattern of polarized mitochondria. The intensity of green mitochondrial fluorescence in cisplatin-treated cells is significantly increased (36.56 to 55.56%) by the co-treatment with ibuprofen. Ibuprofen also promoted the release of cytochrome c from the mitochondria induced by cisplatin (Figure 4b). These findings unequivocally indicated that in A549 cells ibuprofen enhanced the mitochondria-dependent apoptosis caused by cisplatin. Ibuprofen increases the activation of Bax induced by cisplatin The translocation of the pro-apoptotic protein Bax to the mitochondria is closely associated with the apoptosis induced by cisplatin. To explore the mechanisms by which ibuprofen promotes the apoptosis mediated by mitochondria in response to cisplatin we examined whether it was due to its ability to stimulate the translocation of Bax by cisplatin. We first monitored conformational changes in Bax as indicators of its activation. Western blot analysis of the immunoprecipitates with a conformation specific anti-Bax (6A7) antibody which only recognizes the active form revealed the presence of active Bax in A549 cells treated with cisplatin (Figure 5a lane 4) although not in untreated cells (Figure 5a lanes 1 and 2). Further exposure of the cisplatin-treated cells to ibuprofen caused a 1.5-fold increase in active Bax compared with incubation with cisplatin alone (Figure 5a lane 3)."

"BACKGROUND American Indians/Alaskan Natives (AI/ANs) have the worst 5-year cancer survival of all racial/ethnic groups in the United States. Causes for this disparity are unknown. The authors of this report examined the receipt of cancer treatment among AI/AN patients compared with white patients. METHODS This was a retrospective cohort study of 338204 patients who were diagnosed at age ?65 years with breast colon lung or prostate cancer between 1996 and 2005 in the Surveillance Epidemiology and End Results-Medicare database. Nationally accepted guidelines for surgical and adjuvant therapy and surveillance were selected as metrics of optimal guideline-concordant care. Treatment analyses compared AI/ANs with matched whites. RESULTS Across cancer types AI/ANs were less likely to receive optimal cancer treatment and were less likely to undergo surgery (P ? .025 for all cancers). Adjuvant therapy rates were significantly lower for AI/AN patients with breast cancer (P <.001) and colon cancer (P = .001). Rates of post-treatment surveillance also were lower among AI/ANs and were statistically significantly lower for AI/AN patients with breast cancer (P = .002) and prostate cancer (P <.001). Nonreceipt of optimal cancer treatment was associated with significantly worse survival across cancer types. Disease-specific survival for those who did not undergo surgery was significantly lower for patients with breast cancer (hazard ratio [HR] 0.62) colon cancer (HR 0.74) prostate cancer (HR 0.52) and lung cancer (HR 0.36). Survival rates also were significantly lower for those patients who did not receive adjuvant therapy for breast cancer (HR 0.56) colon cancer (HR 0.59) or prostate cancer (HR 0.81; all 95% confidence intervals were <1.0). S Fewer AI/AN patients than white patients received guideline-concordant cancer treatment across the 4 most common cancers. Efforts to explain these differences are critical to improving cancer care and survival for AI/AN patients. American Indian Alaskan Native cancer guidelines treatment PLoS One one 1932-6203 Public Library of Science San Francisco USA 24498395 3912155 PONE-D-13-51055 .0087900 Research Biology Model anisms Animal Models Mouse Molecular Cell Biology Signal Transduction Signaling Cascades c-Jun N-terminal kinase signaling cascade Medicine Oncology Cancer Treatment Gene Therapy Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Anti-Cancer Effects of REIC/Dkk-3-encoding Adenoviral Vector for the Treatment of Non-small Cell Lung Cancer Anti-Cancer Effects of REIC Gene Therapy for NSCLC Shien Kazuhiko 1 2 Tanaka Norimitsu 2 Watanabe Masami 3 4 5 Soh Junichi 2 Sakaguchi Masakiyo 6 Matsuo Keitaro 7 Yamamoto Hiromasa 2 Furukawa Masashi 2 Asano Hiroaki 2 Tsukuda Kazunori 2 Nasu Yasutomo 3 Huh Nam-Ho 6 Miyoshi Shinichiro 2 Kumon Hiromi 3 5 Toyooka Shinichi 1 2 * 1 Department of Clinical Genomic Medicine Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan 2 Department of Thoracic Surgery Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan 3 Department of Urology Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan 4 Center for Gene and Cell Therapy Okayama University Hospital Okayama Japan 5 Innovation Center Okayama for Nanobio-Targeted Therapy (ICONT) Okayama University Okayama Japan 6 Department of Cell Biology Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan 7 Department of Preventive Medicine Kyusyu University Faculty of Medical Science Fukuoka Japan Deb Sumitra Editor Virginia Commonwealth University United States of America * E-mail: toyookamd.okayama-u.ac.jp Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: KS NT MW MS HY MF HA KT YN NHH SM HK ST. Performed the experiments: KS NT. Analyzed the data: KS NT JS KM HY ST. Contributed reagents/materials/analysis tools: KS NT MW MS KM YN NHH HK ST. Wrote the paper: KS NT JS HY ST. 2014 3 2 2014 9 2 e87900 4 12 2013 30 12 2013 2014 Shien et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Objectives REIC/Dkk-3 is down-regulated in a broad range of human cancer cells and is considered to function as a tumor suppressor. We previously reported that REIC/Dkk-3-expressing adenovirus vector (Ad-REIC) induced endoplasmic reticulum (ER) stress and cancer-specific apoptosis in human prostate cancer. In this study we examined the therapeutic impact of Ad-REIC on non-small cell lung cancer (NSCLC). Materials and Methods We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines. Results Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a non-malignant immortalized cell line OUMS-24 was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. "

Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efficacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the final counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artificialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunofluorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image filtering edge detection image expansion and contraction techniquesusing python™s CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and specificity ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and specificityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the œseeds shed from the primary tumor andor metastatic lesions and rooted in a new œsoiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic What™s more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with flow cytometry which is still theœgold standard of CTCdetection Racila Howeverthis method that lack of the cancer specific markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunofluorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes filaments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identification is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identification and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a fluorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression profile Ligthart redefined the CTCs by computer algorithms after the manualcounting The stricter definition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classifier was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classification methods stillremain subjective as the rules are set artificially The fixedconditions may not identify the morphologically heterogeneousCTCs integrally What™s more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artificial intelligence AI recent yearsmachine learning become an exciting field for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the world™s first internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood flow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors™ diagnosis This artificial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the fieldof medical research specifically medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identification Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artificial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during “ which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The first ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical level““testing data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overfittingEnrichment and imFISH Identification ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was first centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared fixed and dried cells were used to perform the imFISHThe slides were fixed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at —¦C The probe was eluted and the slides were washedtwice in — SSC Then the CD45 fluorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at —¦C After incubation CD45 fluorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a fluorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentfluorescent colors Usually manual counting is the œgoldstandard but it™s a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We first selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is — The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image filtering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was defined as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs IdentificationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artificial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasfirst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the first five layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe fivefold cross validation was used to prevent overfittingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The final hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear A“C The imFISH result of CEP8 CD45 and DAPI D The merge of panelsA“C E The CTCs were identified by CV segmentation method and marked in red box a“c The CEP8 signal points were identified by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workflow is shown belowSp The grid was defined on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function œsoftmaxœReLU œtanh kernel regularizer type œl1 œl2regularization factor œ œ The range of possible values were defined of eachdimension Allestablishing the best onethe possibleconfigurations weresearched forEvaluation Criteria for ClassificationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassification model such as sensitivity Se or recall specificitySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 — precision — recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The first five layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix definitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artificial error and save costs we performedthe traditional image identification method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was defined as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1A“C the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identification and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and specificity were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identification of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole flowchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand specificity were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and specificity in CTC countingisslightly worse than the first method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesn™t cover the whole film but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The flowchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspecificity in CTC counting We had changed the filter sizeand number in all convolution layers in order to find thebest CNN parameters We found diï¬erent filter size andnumber will influence the results largely We changed filternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about “ We tried toincrease the filter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the filter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classification Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more influence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened thefluorescence images according to the figure™s quality In order toimprove the efficiency we used the machine instead of ngmanual screening First the python™s package was used to mage segmentation The obtained recognition sensitivity andspecificity are and respectively In addition therecognition sensitivity and specificity can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participants™of kin Written informed consent was obtained from theindividuals and minors™ legal guardiannext of kin for thepublication of any potentially identifiable images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the final version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientific Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers 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"Trophoblast cell surface antigen TROP2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion The association between TROP2 expression and occurrence anddevelopment of oral squamous cell carcinoma OSCC remains to be understoodMethods We investigated the role of TROP2 in OSCC patients using a combination of biophysical approaches Atotal of OSCC patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry KaplanMeier survival curve analysis and atomic force microscopy to analyzeTROP2 expression morphology and mechanical properties of OSCC tissuesResults TROP2 was overexpressed in of poorly differentiated OSCC samples High levels of TROP2 wereassociated with survival rate lower than and patient age odds ratio [OR] P confidence interval [CI “] tumor size OR P CI [“] and TNM stageOR P CI [“] Average surface roughness of low medium and highly differentiatedOSCC tissues were ± ± and ± nm respectively The Pearson coefficient revealed anegative association between tumor stiffness and TROP2 expression r ˆ’ P Conclusion Overexpression of TROP2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics Taken together our findings demonstrated that TROP2 may be an indicator of OSCCdifferentiation leading to the altered mechanical properties of OSCC tissuesKeywords Oral squamous cell carcinoma TROP2 Tissue stiffness Differentiation SurvivalBackgroundOral squamous cell carcinoma OSCC is a commonsubtype of head and neck and other malignant tumors[ ] The past few decades have shown increased incidence of OSCC that is expected to rise further in the future [] Thereforeimperative to determineisit Correspondence zhangkllzu163com Baoping Zhang Shuting Gao and Ruiping Li contributed equally to thiswork1Department Hospital of Stomatology Lanzhou University Donggang westRoad Lanzhou Gansu ChinaFull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of OSCCHuman trophoblast cell surface antigen TROP2 alsocalled tumorassociated calcium signaltransduction2TACSTD2 is a surface glycoprotein encoded by TACSTD that has extracellular domains a single transmembrane domain and a short tail [ ] TROP2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal [] pancreatic [] and laryngealcancers [] Inhibiting TROP2 expression has shownpromise in clinical applications [ ] TROP2 regulates The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Cancer Page of tumorigenic properties including cancer cell adhesion invasion and migration Tang [] have recentlyshown that TROP2 impacts growth and metastasis byactivating PI3KAKT signaling This phenomenon hasalso been observed in gallbladder cancer [] Amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ “] This has shed light on the biological functions of TROP2 and its use as a prognostic biomarker for OSCCvarious biochemical mechanismsAtomic force microscopy AFM is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesAFM has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast [] liver [] and lung [] Parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion[] Advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesThis study aims at investigating the correlation between TROP2 expression and clinicopathological characteristics of OSCC We have demonstrated the tissuemorphology and mechanics of OSCC samples duringtumor development using AFM We believe our findingswill help develop TROP2 in accurately diagnosing OSCCin tumors with different grades of differentiationMethodsTissue preparationThe protocols in this study were approved by the researchethics committee of Lanzhou University Tumor sampleswere collected from patients after obtaining written informed consent A total of patients with oral squamous cell carcinoma OSCC were registered atthesecond hospital of Lanzhou University between January and March Among these samples sampleseach showed high moderate and low levels of differentiation The experimental group comprised males and females aged “ years average years All patientswere diagnosed with OSCC based on surgery andFig Paraffin pathological sections of tissues a d g — 4fold b e h — 10fold c f i — 40fold 0cZhang BMC Cancer Page of Fig Immunohistochemical staining was performed to detect the expression of TROP2 at different stages of OSCCpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery Pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded Cancer and cervical lymph nodetissues were collected after maxillofacial surgery All specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding Among them and Fig Average optical density of TROP2 poorly differentiated squamous cell carcinoma showed high expressionP 0cZhang BMC Cancer Page of Table Correlation between TROP2 expression and clinicopathological characteristicsCharactersnTROP2 expressionLow or noTotalGenderMaleFemaleAge‰¥ LocalizationmucosaTongueDifferentiationwellModeratePoorTumor sizeT1 ‰ cm cmT2 ‰ cmT34cmT4bLymph node metastasesN0NXDistant metastasesM0M1TNM stageI IIIII IVPerineural infiltrationNoYesVascular invasionNoYesPearson x2P value Highpatients exhibited no and cervical lymph node metastasesrespectively Clinical TNM staging was performed according to the 7th edition TNM staging classification standardjointly developed by the International Union for CancerControl and American Joint Committee on Cancer []and World Health anization guidelines []Hematoxylin and eosin HE stainingOSCC tissues were fixed overnight using neutralformalin Solarbio Beijing China paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol The sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing SolarbioBeijing China Sections were visualized and imagedusing the Olympus BX53 at magnifications of — and sealing and neutral 0cZhang BMC Cancer Page of ImmunohistochemistryHE sections were subjected to the SP method to detectTROP2 expression The sections were incubated overnight with the primary antibody against TROP2 Abcam USA at °C followed by incubation withbiotinlabeled goat antirabbitIgG AbcamUSA at °C for h The sections were then developed using DAB Beijing Zhongshan Golden BridgeBiotechnology China dehydrated transparentizationand film and neutral resin sealed The prepared sections were visualized using microscopy OlympusBX53 JapanAFMFixed tissues were placed in Petri dishes containingphosphatebuffered saline All analyses for mechanical properties were performed using the biologicalatomic force microscope BioAFM NanoWizard IIIBruker USA Silicon AFM probes from the Pointprobe®constant of Nmcoating NanoWorld USA wereCONTRreflexused The spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm AFM was performed using theseries with a kHzforcetheresonancefrequencyofcontact model and a scan rate of Hzs in airForcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms [] Six random sites wereselected for each sample and each site was measured times We used the modified Hertz contact modelto analyze forcedistance curves [] and calculateYoung™s modulus and roughness of OSCC tissueswith varying differentiationStatistical analysisStatistical analyses were performed using SPSS Statistical Product and Service Solutions IBM Forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the TukeyKramerHSD test for pairwise comparisons Pearson Chisquaretest was used to analyze clinical features and TROP2 expression based on the calculated odds ratios ORs and confidence intervalCI Survival was evaluatedusing KaplanMeier curves and the difference was analyzed using the logrank test P was consideredstatistically significantFig TROP2 total survival curve using KaplanMeier survival curves low blue line high green line 0cZhang BMC Cancer Page of ResultsTissue morphology and TROP2 expression across theclinical stages of OSCCTumor cells from poorly differentiated OSCC samples exhibited characteristic atypia poor differentiation and irregular morphology Fig Howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated OSCC as compared to those inpoorly differentiated cells TROP2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells We observed that low differentiation and high malignancy of OSCC was associated with higher TROP2 expression Fig Theaverage optical density of TROP2 among the lowmedium and highly differentiated OSCC tissues were ± ± and ± respectively Fig Table TROP2 expression risk factors with clinicopathological featuresCharacteristicsnTROP2 expressionLow or noTotalGenderMaleFemaleAge‰¥ LocalizationMucosaTongueDifferentiationWellModeratePoorTumor sizeT1 ‰ cm cmT2 ‰ cmT34cmT4bLymph node metastasesN0NXDistant metastasesM0M1TNM stageI IIIII IVPerineural infiltrationNoYesVscular invasionNoYesNote a Well vs Moderate b Moderate vs Poor c Well vs PoorP valueOR CIHigh 005a 0001b 0001c 0cZhang BMC Cancer Page of Association between TROP2 expression and clinicalcharacteristics of OSCCWe analyzed the clinicopathological characteristics of patients with OSCC with varying degree of differentiationDifferential expression of TROP2 was associated with patient age tumor differentiation tumor size TNM stagepercutaneous nerveinvasionTable P Patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high TROP2 expressionP However there was no association between theexpression of TROP2 and patient gender tumor locationlymph node metastasis or distant metastases P and vascularfiltrationTROP2 expression and patient survivalUsing KaplanMeier survival curves we observed that anincrease in TROP2 expression negatively correlated withthe overall survival of patients Fig And lowno ofTROP2 expression group™s 3years survival rate was a for high expression group and 5years ratewere and respectively TROP2 expression wasassociated with patient age P OR CI[“] tumor differentiation Well vs ModerateP OR CI [“] Moderate vsPoor P OR CI [“]Well vs Poor P OR CI [“] tumor size P OR CI[“] TNM stage P OR CI[“] vascular invasion P OR CI [“] and peripheral nerve invasionP OR Table High TROP2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher TNM staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival Thus TROP2 may be a prognostic indicator for survival in OSCC patientsFig Surface morphology of OSCC tissue sections via AFM detection irregular morphology appeared in the low differentiation 0cZhang BMC Cancer Page of Surface morphology and roughness of OSCC tissuesThe surface morphologies of OSCC tissues with varying degrees of differentiation were analyzed directtopographical imaging using BioAFM Figure showsthe representative image from each tissue acquiredduring the cantileverbased AFM nano indentationtest The tissue interface varied with tumor differentiationindicating that highly differentiated OSCC tissues had a regular and flat morphology OSCC tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization Figure summarizes the roughness ofOSCC tissues with varying differentiation The average surface roughness of low medium and highly differentiated OSCC tissues were ± ± and ± nm respectively Roughness ofthe tissue surface was enhanced with increasing differentiation of OSCC tissuesYoung™s modulus of OSCC tissuesWe used BioAFM to determine Young™s modulusbased on the mechanical properties of OSCC tissues with varying degrees of differentiation We randomly selected six contact points from each slice andeach contact point was measured times Forcedistance curves were generated for each slice and theJPK Data Processing software version was usedto calculate Young™s modulus Figure shows theaverage variation in stiffness within individual tissuesin the range of “ kPa In the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples P Fig Surface roughness results are express as mean ± SEM nm 0cZhang BMC Cancer Page of Fig AFM test average tissue stiffness Young™s modulus E was thus confirmed to be a parameter of cell hardness for various cells and tissuePa P Thus tissue differentiation was positively associated with its stiffness Fig Association between mechanical properties and TROP2expression in OSCCThe Pearson coefficient showed a negative associationbetween the stiffness of OSCC tissues and TROP2 expression Fig r ˆ’ P Thus we detectedan increase in stiffness with varying differentiation in thetumor samplesDiscussionTROP2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines Studieshave shown that overexpression of TROP2 is associatedwith tumorigenesis and malignancy [“]In thisstudy TROP2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness The relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaImmunohistochemical analysis revealed that the expression of TROP2 in poorly differentiated OSCC tissueswas significantly higher than that in welldifferentiatedOSCC tissues Additionally TROP2 upregulation wascorrelated with tumors of advanced TNM III IV staging and poor differentiation than that in tumors withlow TNM I II staging Thus the abnormal expressionof TROP2 may be associated with the occurrence anddevelopment of tongue malignancies Furthermore highTROP2 expression predicted low survival as comparedto that in the tumors with low TROP2 expression Previous research has also demonstrated the correlation between shorter patient lifespan and high levels of TROP2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of TROP2 [] TROP2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis[] Upregulated TROP2 correlates with the poor prognosis of thyroid papillary carcinoma [] colon cancer[] liver cancer [] and other malignanciesThere have been an increasing number of studies on thebiological role of TROP2 at the molecular level TROP2induces the downregulationloss of PTEN thereby stimulating PI3KAKT signaling and tumor development []PTEN is a wellknown tumor suppressor that is a phosphatase [] and affects the PI3KPKBAKT signaling axisduring the dephosphorylation of PIP2 and PIP3 []PI3K signaling is important in regulating tumor cell proliferation migration and invasion [ ] Thus PTEN is anegative regulator of cancer [ ] Li have shownthat TROP2 activates epithelialmesenchymal transitionvia PI3KAKT signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer [] Similarly TROP2 expression stimulates the proliferation migration and invasion of osteosarcoma cells [] Hou et aldemonstrated that TROP2 regulates JAK2STAT3 signaling in glioblastoma cells [] 0cZhang BMC Cancer Page of Fig Correlation analysis between changes in mechanical stiffness of OSCC tissues and TROP2 expression Note changes have statisticalsignificance P and show a certain negative correlation r ˆ’ Functional differentiation oftissues influences themicromorphology and mechanical stiffness of OSCCcells We detected low surface roughness on OSCC tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion Poorlydifferentiated OSCC tissues are œsofter than highly differentiated OSCC tissues PI3K is an important celladhesion molecule TROP2 triggers the synthesis of proteins with homologous domains such as pleckstrinRAC Tiam and Vav Tiam and Vav activate RAC thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion []The underlying mechanisms involved in the alterationof micromechanical properties of OSCC samples and occurrence development metastasis and invasion ofOSCC tumors remain to be elucidated HE staining isthe gold standard for tumor diagnosis With the development of biomechanics in the past two decades [] the mechanical properties of tissues need to be investigated based on biomedical and physical parametersIn this study we have assayed the changes in mechanicalproperties at the micronanometer level using AFM anddetermined the association between the TNM grademetastasis and stiffness of tumor samplesIn conclusion we have demonstrated the association between differential expression of TROP2 and patient agetumor differentiation tumor size TNM stage percutaneousnerve filtration and vascular invasion Moreover high levelsof TROP2 correlated with poor overall survival in patientsHighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness Lastly high TROP2 expression resulted in reduced tumor stiffness However thisstudy had some limitations First the cohort used in thisstudy was relatively small Second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay Thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developTROP2 as a specific and efficient prognostic biomarker forOSCCConclusionThese findings could promote new methods for the earlyOSCC diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity More detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseAbbreviationsOSCC Oral squamous cell carcinoma TROP2 Trophoblast cell surfaceantigen AFM Atomic force microscopyAcknowledgementsWe thank the individual who participated in this studyAuthors™ contributionsBZ SG and RPL are responsible for conception and design Data wascollected by YTL RC JYC and YMG Data was analyzed by EW and YH KLZrevised the All authors have read and approved the manuscriptFundingThis work was supported by the Fundamental Research Funds for theCentral Universities No lzujbky2020cd03 Baoping Zhang Doctoralmaster 0cZhang BMC Cancer Page of students of the Second Hospital of Lanzhou University sdkygg17 Lan Yangand Key Laboratory of Mechanics on Disaster and Environment in WesternChina The Ministry of Education of China No “ Kailiang ZhangAvailability of data and materialsThe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateWritten informed consent was obtained from each participant before samplecollection The study was approved by the Committee for Ethical Affairs ofSchool of Stomatology Lanzhou UniversityConsent for publicationNot applicableCompeting interestsThe authors have no conflicts of interestAuthor details1Department Hospital of Stomatology Lanzhou University Donggang westRoad Lanzhou Gansu China 2Institute of Biomechanics andMedical Engineering Lanzhou University Lanzhou ChinaReceived April Accepted August ReferencesIyer S Thankappan K Balasubramanian D Early detection of oral cancerscurrent status and future prospects Curr Opin Otolaryngol Head Neck Surg“Caldeira PC Soto AML de Aguiar MCF Martins CC Tumor depth of invasionand prognosis of earlystage oral squamous cell carcinoma a metaanalysisOral Dis Online ahead of printKim Y Kim JH Increasing incidence and improving survival of oral tonguesquamous cell carcinoma Sci Rep McDougall AR Tolcos M Hooper SB Cole TJ Wallace MJ Wallace Trop2from development to disease Dev Dyn “Guan GF Zhang DJ Wen LJ Yu DJ Zhao Y Zhu L Prognostic value ofTROP2 in human nasopharyngeal carcinoma Int J Clin Exp Pathol “Stewart D Cristea M Antibodydrug conjugates for ovarian cancer currentclinical development Curr Opin Obstet Gynecol “Liu J Yang D Yin Z Gao M Tong H Su Y A novel human 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for latestage epithelial carcinomas Biochim Biophys Acta “ Zargari N Mokhtari M Evaluation of diagnostic utility ofimmunohistochemistry markers of TROP2 and HBME1 in the diagnosis ofthyroid carcinoma Eur Thyroid J “ Zhao P Zhang Z TNFα promotes colon cancer cell migration and invasionby upregulating TROP2 Oncol Lett “Sin STK Li Y Liu M Yuan YF Ma S Guan XY Downregulation of TROP2predicts poor prognosis of hepatocellular carcinoma patients HepatolCommun “ Zhang Y Zhang R Luo G Ai K Long noncoding RNA SNHG1 promotes cellproliferation through PI3KAKT signaling pathway in pancreatic ductaladenocarcinoma J Cancer “Sai J Owens P Novitskiy SV Hawkins OE Vilgelm AE Yang J PI3Kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antiPD1 responses Clin Cancer Res “ Chen X Pang B Liang Y Xu SC Xin T Fan HT Overexpression of Zhang XR Wang SY Sun W Wei C Isoliquiritigenin inhibits proliferation andEpCAM and Trop2 in pituitary adenomas Int J Clin Exp Pathol “metastasis of MKN28 gastric cancer cells by suppressing the PI3KAKTmTOR signaling pathway Mol Med Rep “ 0cZhang BMC Cancer Page of Wise HM Hermida MA Leslie NR Prostate cancer PI3K PTEN and prognosisClin Sci Lond “ Yuan B Zou M Zhao Y Zhang K Sun Y Peng X Upregulation of miR130b3p activates the PTENPI3KAKTNFκB pathway to defense againstmycoplasma gallisepticum HS Strain infection of chicken Int J Mol Sci pii E2172Li JW Wang XY Zhang X Gao L Wang LF Yin XH Epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of thePTENPI3KAKT pathway Mol Med Rep “Li X Teng S Zhang Y Zhang W Zhang X Xu K TROP2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating PI3KAKT pathway and inducing EMT Oncotarget “ Gu QZ Nijiati A Gao X Tao KL Li CD Fan XP TROP2 promotes cellproliferation and migration in osteosarcoma through PI3KAKT signalingMol Med Rep “ Hou J Lv A Deng Q Zhang G Hu X Cui H TROP2 promotes theproliferation and metastasis of glioblastoma cells by activating the JAK2STAT3 signaling pathway Oncol Rep “ Rivard N Phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function Front Biosci Landmark Ed “ Pankova D Jiang Y Chatzifrangkeskou M Vendrell I Buzzelli J Ryan A et alRASSF1A controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma EMBO J 20193813e100532 Wullkopf L West AV Leijnse N Cox TR Madsen CD Oddershede LB et alCancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential Mol Biol Cell “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ‰¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ‰¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ‰¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ “ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinson™s and angina κ “ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworld™s older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ‰¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV “ two algorithms which determine an individual™scurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [“] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ‰¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ‰¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ‰¥ years The first wave ofdata collection began in October through toFebruary N participants aged ‰¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDA™s study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ‰¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ‰¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ‰¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participant™s TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinson™s disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity “ Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ‰¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system [“]Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question eg˜donepezil™ was required to be dispensed on ‰¥ consecutive prescriptions to link this medication withthe RxRiskV condition ˜dementia™ This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ‰¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions “ eg antiwere notthrombotic agents B01AC04 “ B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ‰¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohen™s Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair “ moderate “ good “ and verygood “ []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ‰¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ‰¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ‰¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ‰¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ‰¥ years N Poor delayed recall years N ‰¥ years N years N ‰¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients™ selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ‰¥ years measures of morbidity High blood pressure or hypertension yearsN ‰¥ years N and highcholesterol years N ‰¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ‰¥ years years N “Age‰¥ years N “GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinson™s κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ “ All other conditions had poor agreement κ “ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was ˜very good™ agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe ˜very good™ Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinson™s disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was ˜good™ agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was ˜moderate™ agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was also˜moderate™ agreement for high cholesterol in both agecohorts and for angina and Parkinson™s disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ‰¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPaget™s disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinson™s disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack “ “StrokeTIAHigh Cholesterol “ “ “HeartTrouble “Cancer “EmotionalProblems “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Depressiononly “ “ “ “ “ “Stomachulcers “ “ “ “ “ “Asthma “ “ “ “ “ “Arthritisgeneral “ “ “ “ “ “RheumatoidArthritis only “ “ “ “ “ “ “ “ “Angina “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Congestive HeartFailure “Abnormal HeartRhythm “ “ “ “ ““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““ “ “ “““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Anxiety “ “ “ “ “ “ “ “ “ “LungDisease “ “ “ “ “ “ “ “ “ “ 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis “Psychosisonly “StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinson™s disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinson™s diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only ˜fair™ agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso ˜fair™ agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified ˜taking pain medication™ Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was œpoor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also ˜poor™ agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed ˜diapers and pads supplies™ []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo

" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi

predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior “ which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients™ responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief model™s components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zˆd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbach™s alphafor face validity a survey was done on “ employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tool™s questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbach™s alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartlett™s research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedon™t know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring range˜correct™ response ˜don™t know™response ˜incorrect™ response “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “ perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individual™s perception of his orher competence to successfully perform a behavior behavioral intention refers to a person™s perceived probability orœsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “ behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items χ2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartlett™s test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range “ if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of education““femalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop “value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ˆ’ ± ± ± ± ˆ’ ± ± ˆ’ ± ˆ’ ± ± ± ˆ’ ± ± ˆ’ ± ± ˆ’ ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that one™sintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the œresearcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors™ contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth “ roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh “ alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j “ braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med “ gimenogarc­a az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol ““ bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol “luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol “ ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci “ cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarc­a r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health “ becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health “ ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol “ moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med“ rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures “available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh “centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239“ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health “hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran “ anz j surg “yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev “ glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis “ becker mh the health belief model and sick role behavior health educmonogr “janz n champion v strecher vj the health belief model k glanz bk rimer“janz nk becker mh the health belief model a decade later health educ q“lp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav“ hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c"

microbiota involves communities ofhepatitis is generally known as an ‚ammation of the liver that can be caused by hepaticand nonhepatic viruses can be caused by alcohol can be drug induced and can be caused byautoimmunity gut microbiota composition is known to be associated with disease pathogenesishowever dynamic alteration of the gut microbiota in disease pathogenesis is not wellunderstoodsymbiotic as well as pathogenicmicroanisms found in anisms ie plants and animals microbiota of a healthy individualshows more of commensalism or symbiosis without causing any disease these microbes mainlycolonize humans during birth or shortly thereafter and remain throughout the course of life thesecan be found in many areas like skin respiratory tract urinary tract and digestive tract whilebrain lungs and the circulatory system are free of microbes approximately microbes arepresent in a healthy individual gut minemura and shimizu therefore gut microbiota hasan important role to modulate the immune system in disease progression or recoverycommensaltranslocation of microbes or their metabolic products cause intestinal ‚ammation leadingto impairment of the primary barrier hill there is limited available informationregarding the role of gut microbiota in hepatitis which makes it important to majorly focus onclinical data of gut microbiota linked with hepatitis b and c virusgut microbiotagut or gastrointestinal tract starts from the mouth and ends at the back passage anus gut helpsin the digestion of food by absorbing energy and nutrients majority of gut microbiota to contains good bacteria and only to are harmful bacteria in diï¬erent parts of the intestineedited bymilan surjittranslational health science andtechnology institute thsti indiareviewed byjawed iqbaljamia millia islamia indiabinod kumarloyola university chicagounited statescorrespondencenirupma trehanpatitrehanpatigmailcomspecialty sectionthis was submitted tovirus and hosta section of the frontiers in cellular and infectionmicrobiologyreceived march accepted june published august citationsehgal r bedi o and trehanpati n role of microbiota inpathogenesis and management ofviral hepatitisfront cell infect microbiol 103389fcimb202000341frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesbajaj in mouth and upper respiratory tract normalflora is more of the commensal bacteria like streptococcusmoraxella neisseria and haemophilus very few species ofbacteria are present in the stomach and small intestine whilethe large intestine and colon contain dense population ofmicrobes ie up to cellsg along with bacteria many othermicroanisms like fungi protists archaea and viruses alsosymbiotically harbor in the gutthere are four dominant phyla of bacteria present in thegut and they are firmicutes bacteroidetes actinobacteriaand proteobacteria khanna and tosh most importantgenera in which bacteria belong are bacteroides clostridiumfaecalibacterium eubacterium ruminococcus peptococcuspeptostreptococcus and bifidobacterium fern¡ndez some of the fungal species that also coexist in the gut arecandida saccharomyces aspergillus penicillium rhodotorulatrametes pleospora sclerotinia bullera and galactomycesamong others raimondi functions of gut microbiotagut microbiota plays an important but diverse role such asbarrier eï¬ect vitamin synthesis and fermentation residentbacteria of the gut acts as a barrier and protect the intestinalmucosa from invasion of the other potential pathogens hooper many factors including diet age medicationillness stress and lifestyle ‚uence the gut microbiota whichhave a great impact on disease pathogenesis in fact manybacteria ie bacteroides eubacterium propionibacterium andfusobacterium are instrumental in the synthesis of vitamins kand b ie folate b12 and biotin canny and mccormick they are also involved in the fermentation of nondigestible carbohydrates for the production of shortchain fattyacids scfas which are helpfulin maintaining metabolichomeostasis in addition to the production of scfa glycolysisand pentose phosphate pathway also produce butyrate whichpromotes the growth of lactobacilli and bifidobacteria bacteriain the colon venegas various studies supported thefact that nutrients derived from microbiota play a pivotal rolein the normal functioning of the hepatic system li zheng moratalla jiminez cremer wang 2017agut microbiota in liver diseasescommensal bacteria play a decisive role in maintaining immunehomeostasis figure and also guard immune reactions atmucosal surfaces ichinohe intestinal microflora isa dynamic and complex ecosystem which helps in proliferationgrowth and diï¬erentiation of epithelial cells to fight infectionsand improve immunity despite its crucial role in the synthesisfolate scfa and peroxides gut microbiotaof vitamin kacts as a chief environmental as well as etiologicalfactorfor the progression of many liver diseaseso™hara andshanahan particularly gut microbiota has a larger‚uence on alcoholic liver disease nonalcoholic fatty liverdisease viral hepatitishepatitis b and c autoimmunehepatitis aih primary sclerosing cholangitis psc andprimary biliary cholangitismohamadkhani pbclactobacillus bifidobacterium saccharomyces boulardii andlactobacillus plantarum play a bigger role in the managementof various metabolic disorders and hepatitis mohamadkhaniincludingvirusesandseveralpathogensintestinalmicroanisms use mucous membranes as a doorwaykarst hepatic viruses breach the intestinal permeabilityleading to gut dysbiosis and release pro‚ammatory cytokinesinstrumental in developing liver cirrhosis and hcc it is alsoobserved that the use of probiotics reduces the tolerogenicresponse and enhances the mucosal defense against viralpathogens rigoadrover m del lactobacillusalone can ‚uence the production of interferon by modulatingthe antiviral eï¬ects of vitamin a lee and ko themixture of various probiotics and bifidobacterium with galactooligosaccharides and fructooligosaccharides has a defensiveeï¬ect against rotavirus infection by aggregating the productionof tnfα il4 ifnÎ and tlr2 expression rigoadrover mdel in most of the liver disease especially cirrhosisdysbiosis of the gut increases proteobacteria enterobacteriaceaeand veillonellaceae while it decreases bacteroidetes andlachnospiraceae sanduzzi zamparelli recentlythe cirrhosis dysbiosis ratio cdr is coined for definingthe changes in gut microbiome in cirrhosis patients withbeneficial lachnospiraceae and ruminococcaceae and harmfulenterobacteriaceae bacteria bajaj other groupshave also associated patients with severe cirrhosis and hepaticencephalopathy with overgrowth of enterobacteriaceae bacteriachen role of gut microbiota in hepaticviral infectionsacute viral hepatitis due to hepatitis a and e viral infectionsis a major community health problem especially in developingcountries hepatitis a and e cause acute infection whichcould be shortlived and selfclearing unless the subjects areimmunocompromised or in transplant settings acute hepatitise infection also becomes detrimental and lifethreatening duringpregnancy aï¬ecting both the mother and the childboth hepatitis a and e are rna viruses thattransmitthrough oral fecal routes lemon and may havedevastating eï¬ects on intestinal microflora it was observedthat administration of the healthy probiotic bacterium likeenterococcus faecium ncimb aï¬ects the reduction as wellas the removal of enteric hev viruses in pigs kreuzer however there is lack of relevant data in humansas per the world health anization who hepatitisb virus hbv infection caused deaths in and million diagnosed with chronic infection in similarly hepatitis c virus hcv caused deaths with anestimated million diagnosed with chronic infection in both these viruses cause chronic infections at in hbv andmore than in hcv leading to cirrhosis and hepatocellularcarcinoma hepatic viruses have evolved mechanisms to avoidtheir detection from the host innate and adaptive immunityfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesfigure protective role of fecal microbiota transplantation and use of probiotics in immune restorationand characterized as viral escape visvanathan itis observed that chronic hepatitis patients have largertranslocation of the intestinal microbiota lu li bacterialtranslocation cause intestinal‚ammation viadysregulation of immune cell overgrowth of pathogenic bacteriaas well as dysfunction of the primary barrier hill xu also supported the fact that intestinal floraloses homeostasis during dysbiosis which in fact helps theadvancement of hepatitis viral infection xu itthereforeis now understood that during chronicitycommensal microbiota have greater impact not only on viral hostcell interaction but also on viral replicationin viral hepatitis few harmful bacteria like escherichia colienterobacteriaceae enterococcus faecalis and faecalibacteriumprausnitzii directly alter the profile of good intestinal microbiotawith a lower number of intestinallactic acid species suchas lactobacillus pediococcus weissella and leuconostoc bajaj chen some of the bacterial species ieneisseria e coli enterobacteriaceae e faecalis f prausnitziiand gemella are also found responsible for the progression ofhepatitis b and c virus“related cirrhosis and primary biliarycirrhosis chen mohamadkhani candidais also frequently found in patients with hepatitis b“relatedcirrhosis cui role of gut microbiota in hepatitis b viralinfectiondysbiosis of gut microbiota in chronic hepatitis b infectionaï¬ects disease pathogenesis and causes liver failure in alarge proportion lps lipopolysaccharides from the outermembrane of gramnegative bacteria help in the activationof innate immune response by recognizing tlrs especiallytlr2 and hbv infection leads to progressive declinein butyrateproducing bacteria however lpsproducinggenera is enriched in hbv infection in hbv infection abeneficial bacterium lachnospiraceae plays a role in themanagement of hbv infection via reduction in lps sectionand bacterialtranslocation chen ren studies have shown the role of faecalibacteriumpseudobutyrivibrioruminoclostridiumprevotella alloprevotella and phascolarctobacterium in potentialanti‚ammatory scfa activity which increases the abundanceof butyrate compared to normal subjects liu lu have demonstrated that copy numbers of fprausnitzii e faecalis enterobacteriaceae bifidobacteria andlactic acid bacteria lactobacillus pediococcus leuconostocand weissella have marked variation in the intestine of hbvcirrhotic patients during hbv infection dysbiosis in theoral microbiota was observed and yellow tongue coating issuggestive of a reduction in bacteroidetes but an increaselachnoclostridiumfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesin proteobacteria zhao also suggested positivecorrelation of neisseriaceae with the serum hbvdnacirrhotic patients with hbv infection showed a significantdecrease in the bifidobacteriaceaeenterobacteriaceae be ratiolu while yun observed no diï¬erence in thebe ratio in hbsag with normal or high alt and in noncirrhotic hbv carriers yun it means the be ratiois disturbed only in cirrhosis however other study observedthat the megasphaera genus from the firmicutes phylum wasabundant in the hbsag high alt group than the normal altin patients with normal alt butyrateproducing bacteria likeanaerostipes are more in feces compared to hbsagve yun it is interesting to note that both megasphaeraand anaerostipes produce scfa as a byproduct of lactatefermentation and butyrate however butyrate is known asanticarcinogenic and anti‚ammatory and plays a role inoxidative stress hamer another study suggests thatchronic hepatitis b infected cirrhotic patients exhibit a decreasein bifidobacteria and lactobacillus levels while significantlyincreasing enterococcus and enterobacteriaceae levels comparedto healthy individualsbacterial translocation is also observed in the developmentof hepatocellular carcinoma hcc recently wang havedefined the serum zonulin as an intestinal permeability markerand showed its association with afp levels in hbvassociatedliver cirrhosis and hcc they are helpful in correlating it withadvanced stages of the diseases fasano the use of probiotic in hbvinfected patients showed benefitand suggested that probiotic vsl3 plays an important role in themanagement of hbv viral infection dhiman role of gut microbiota in hepatitis c viralinfectionchronic hepatitis c infection is another leading cause ofcirrhosis hcc and in some casesliver failure and deathin majority enterobacteriaceae and bacterioidetes increased inchronic hcv patients but firmicutes found to be decreasedhcv infection cause marked elevation in lps which issuggestive of microbial translocation and ‚ammation duringdisease progression dolganiuc inoue on the other hand it was observed that antiviral treatment ofhcv with ribavirin rbv and immune modulator pegylatedinterferon pegifn has no direct impact on gut dysbiosisin factit increases the production of bile acids which isimportant for gut microbiota ponziani somepathogenic bacteria such as enterobacteriaceae staphylococcusand enterococcus decreased the bile acid in hcvinfectedcirrhotic patients which normalized after a directacting antiviraltreatment oral directacting antivirals daas were also foundto be helpful in improving gut especially lachnospira and doreagenera and restored tnfα levels p©rezmatute but after daa treatment expression of calprotectin zo1and lps was found more in hcv patients with cirrhosis itwas also suggested that during hcv infection l acidophilusand bifidobacterium spp can act as a supportive supplementwith antiviral and antibacterial activities dore immune response in hcv patients can be stimulated by usefulmicrobiota via activation of cd3 cells and cd56 nk cellcounts which were explained by doskali and furthersuggested that good flora increases the cytotoxic eï¬ects of nkcells against viral infected cells inhibiting the replication of hcvuse of probiotics in hcvinfected patients with cirrhosis wassignificantly beneficial preveden another hepatic virus hepatitis d virus is a new playerand not much is known about it yet it was also suggestedthat endotoxemia in hcv and hdv patientstobe multifactoriallikely depending on impaired phagocyticfunctions and reduced tcellmediated antibacterial activitykefalakes and rehermann seemsmicrobiota modulates molecularsignaling in hepatitisactivereceptorthe keycomponent of gramnegative bacterialps isie enterobacteriaceae thefor lps iscd14tlr4md2 receptor complex on induction whichsecretes many pro‚ammatory cytokines including tumornecrosis factorα il1 il6 and chemokines through the nfκbsignaling fooladi seki and schnabl bryant to cause liver injury in the intestinal tract lpsdownregulates the expression of various tight junction proteinszo1 and closed protein by increasing the permeability ofthe intestinal mucosa and enters the blood flow through theportal venous system park in liver kupï¬er cells asspecialized macrophages are induced by the lpstlr4 pathwayfor the release of immunosuppressive mediators such as il10which in turn suppress the release of ‚ammatory mediatorsby kupï¬er cells dixon in this way during viralhepatitis virus specific immune responses are suppressed andultimately inhibit efficient clearing of bacteria as well as virusesin addition to lps unmethylated cpg dna bacterialdnarna bacterial cell wall also contains teichoic acidpeptidoglycan and specialized proteins flagellin bacterialdnarna is recognized by tlrs as well as all components ofcellwalllike teichoic acid and peptidoglycan also recognized bytlr2 while tlr5 got activated by flagellin dsrna bacteriaare recognized by tlr3 ssrna activates receptors of bothtlr7 and tlr8 all these tlrs ultimately stimulate the jakstat pathway hepatitis viruses are also recognized by tlrs inthe liver or in the intestine and activate downstream signalingpathways mencin unmethylated cpg dnas are found abundantly in thelactobacillus family ie l casei l plantarum l rhamnosus andothers like bifidobacteria proteobacteria and bacteroidetes inthe intestinal flora of animals unmethylated cpg dna is sensedby tlr9 expressed on various mononuclear cells and stimulatesboth innate immune response as well as adaptive immuneresponse krieg kauppila activation the ofcpgtlr9 pathway stimulates downstream molecules of myd88such as irak4 traf6 and irak1 ultimately triggering nfκb and mapk signaling pathways these downstream pathwayshelp in the activation of dcs for the secretion of cytokines andfrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0cfrontiersincellluarandifnectionmcroboogyiilwwwfrontiersniaugustlvoumearticeltable randomized fmt clinical trials for the treatment of chronic hepatitis b infectionsnostudy titlestudy typeno ofsubjectsinterventiontreatmentstatusphaseprimary outcomemeasuresrandomized controlled trialcomparing the efficacy andsafety of fmt in hepatitis breactivation leads to acuteon chronic liver failurelocationinstitute of liver and biliarysciencesnew delhi delhi indiastudy on effect of intestinalmicrobiota transplantationin chronic hepatitis blocation zhongshanhospital affiliated to xiamenuniversityxiamen fujian chinainterventionalclinical trialdrug tenofovirdrug fecal microbiotatransplantation fmtcompletedcompletedtransplant free survival[time frame months]interventionalclinical trialother intestinalmicrobiota transplantdrug antiviral agentsrecruitingnachange of serum hepatitis bvirus e antigenhbeag level[time frame months]serum hepatitis b virus eantigenhbeag levels ismeasured in scosecondary outcome measuresclinicaltrialsgovsehgaletalidentifiernct02689245nct03429439reduction in hepatitis b virus dnalevel ‰¥ log [time frame weeks]improvement in meld model forend stage liver disease score[time frame weeks]change of serum hepatitis b virussurface antigenhbsag level [timeframe months] serumhepatitis b virus surfaceantigenhbsag levels is measuredin iumlchange of serum antihepatitis bvirus e antigenantihbe [timeframe months] appearanceof serum antihepatitis b virus eantigenantihbe suggest the abilityof body to resistant hbvchange of serum antihepatitis bvirus surface antigenantihbs[time frame months]appearance of serum antihepatitisb virus surface antigenantihbssuggest the ability of body toresistant hbvchanges of gut microbiota [timeframe months] alpha andbeta diversity of gi microbiota byhighthroughput sequencing 16srrna on baseline line and months after treatmentrelief of constipation [time frame months] relief of diarrhea[time frame months] reliefof abdominal pain [time frame months] the onset andduration of constipation will beassessed by œevaluation scoretable of gastrointestinalsymptomsimcrobotaiandlveriidseases 0csehgal microbiota and liver diseaseschemokines krieg kauppila chronic hbvpatients have reduced lactobacillus and bifidobacteria both arerich in unmethylated cpg dna levels ultimately aï¬ecting thecpg dnatlr9 pathway and immune response on hbv linand zhang role of fecal microbialtransplantation fmt in viralhepatitisfmt mainly involves the insertion of healthy microbiota in thediseased gut in brief fecal matter derived from a healthy familymember of the patient receiving the same diet as the patient isprocessed and introduced in the intestinal tract of the patientthese have minimal side eï¬ects and proved helpful in reinstatinghealthy gut flora in the patient fmt administration can bedone using several routes such as oral nasogastric nasoduodenalnasojejunal endoscopic rectal and colonoscopic or midguttransendoscopic enteral tubing cui tang for cirrhotic patients with dysbiosis small bowel route ismost aï¬ected while mostly used route is oral delivery in severealcoholic hepatitis sah in comparison to steroids fmt isassociated with decreased disease severity and improved survivalearlier wang 2017b have observed that fmt restored thecognitive function liver function indexes and tlr response incarbon tetrachloride ccl4induced acute hepatitis in ratswoodhouse have observed in a profit clinical trialthe benefits of fecal microbiota transplantation in the smallbowel of cirrhotic patients woodhouse meiglani also observed that cirrhotic patients with antibioticresistant clostridioides difficile infection cdi responded wellafter fmt treatment in factfecal microbiota of alcoholresistant mice when given to alcoholsensitive mice has reducedbacteroidetes and increased actinobacteria as well as firmicutesand protected steatosis development ferrere limited studies are published yet on fmt administration inalcoholrelated liver disease however all these studies showedimmense benefit of fmt bajaj observed the recoveryof cognitive function and hepatic encephalopathy in patientsunder clinical trial after administration of fmt studies recentlypublished from our center have found better efficiency of fmtreferencesbajajj s heuman d m hylemon p b sanyal a the cirrhosis dysbiosisb monteith p changescomplicationsin the gut microbiomej hepatolassociated with cirrhosis“j white mratio definesand its101016jjhep2013bajaj j s kassam z fagan a gavis e a liu e cox i j fecal microbiota transplant from a rational stool donor improveshepatic encephalopathy a randomized clinical trial hepatology “ 101002hep29306bryant c e symmons m and gay n j tolllike receptor signallingthrough macromolecular protein complexes mol immunol “ 101016jmolimm201406033in severe alcoholic patients than standard medical treatmentsarin there are only a couple of randomizedfmt clinical trials for chronic hepatitis b infected patientstable recently groups have addressed how fmt is modulatingimmunity in gut and liver mucosaassociated invariant tmait cells are found abundant in liver to ofintrahepatic t cells gut peripheral blood as well as lungs gao have observed that functional mait cells were altered insah resulting in more bacterial infection in patients alterationin circulating mait cells is observed with defective antibacterialcytokinecytotoxic response against the infection gao they believe that fmt administration has a profoundeï¬ect on the expression of mait cells in alcoholrelated diseasessummary and conclusionlikeruminoclostridiumgut microbiota has an important role in viral alcoholicand metabolic liver diseases gut microbiota plays a crucialrole in modulating the tolllike receptors nfκb signalingjanus kinasesignal transducer and transcription jakstatpathway and cd4t cell activation numerous usefulmicrobiotasfaecalibacteriumlachnoclostridium prevotella alloprevotella pseudobutyrivibrioand phascolarctobacterium play an important role in potentiatinganti‚ammatory short chain fatty acid scfa activity andincreased the butyrate abundance which play a crucial role inthe management of various hepatitisrelated viral infectionsfecal microbiota transplantation became an attractive andsafest mode of treatment for the management of various liverdiseases especially in severe alcoholic hepatitis despite recentpublications there are still gaps in understanding the role ofmicrobiota in viral hepatitis especially in acute hav and hevviral infections therefore there is a need to explore more inthese infectionsauthor contributionsrs and ob written the review nt provide valuablesuggestions corrected and revised all authors contributed to the and approved the submitted versioncanny g o and mccormick b a bacteria in the intestine helpfulresidents or enemies from within infection and immunity am soc microbiol “ 101128iai0018708chen y ji f guo j shi d fang d and li l dysbiosis of smallintestinal microbiota in liver cirrhosis and its association with etiology sci rep 101038srep34055chen y yang f lu h wang b chen y lei d characterization of fecal microbial communities in patients with liver cirrhosishepatology “ 101002hep24423cremer j arnoldini m and hwa t eï¬ect of water flow and chemicalenvironment on microbiota growth and composition in the human colon procnatl acad sci usa “ 101073pnas1619598114cui b feng q wang h wang m peng z li p fecalmicrobiota transplantation through midgut for refractory c rohn™s diseasefrontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasessafety feasibility and efficacy trial results j gastroenterol hepatol “ 101111jgh12727cui l morris a and ghedin e the human mycobiome in health anddisease genome med 101186gm467dhiman r k rana b agrawal s garg a chopra m thumburu k k probiotic vsl\\ reduces liver disease severity and hospitalization inpatients with cirrhosis a randomized controlled trial gastroenterology “ 101053jgastro201408031dixon l j barnes m tang h pritchard m t and nagy l e kupï¬ercells in the liver compr physiol “ 101002cphyc120026dolganiuc a norkina o kodys k catalano d bakis g marshall c viral and host factors induce macrophage activation and loss of tolllikereceptor tolerance in chronic hcv infection gastroenterology “ 101053jgastro200708003dore g ward j and thursz m hepatitis c disease burden andstrategies to manage the burden guest editors mark thursz gregory doreand john ward j viral hepat 21suppl “ 101111jvh12253doskali m tanaka y ohira m ishiyama k tashiro h chayama k possibility of adoptive immunotherapy with peripheral bloodderived cd3ˆ’cd56 and cd3cd56 cells for inducing antihepatocellularcarcinoma and antihepatitis c virus activity j immunother “ 101097cji0b013e3182048c4efasano a intestinal permeability and its regulation by zonulin diagnosticand therapeutic implications clin gastroenterol hepatol “ 101016jcgh201208012fern¡ndez m f reinap©rez i astaj m rodriguezcarrillo aplazadiaz j and fontana l breast cancer and its relationshipwith the microbiotaj environ res public health int 103390ijerph15081747ferrere g wrzosek l cailleux f turpin w puchois v spatz m fecal microbiota manipulation prevents dysbiosis and alcoholinduced liver injury in mice j hepatol “ 101016jjhep2016fooladi a i tavakoli h and naderi a detection of enterotoxigenicjin domestic dairy productsisolatesiranstaphylococcus aureusmicrobiol gao b ma j and xiang x mait cells a novel therapeutic target foralcoholic liver disease gut “ 101136gutjnl2017315284hamer h m jonkers d venema k vanhoutvin s troost f and brummerr j the role of butyrate on colonic function aliment pharmacol ther “ 101111j13652036200703562xhill d a hoï¬mann c abt m c du y kobuley d kirn t j metagenomic analyses reveal antibioticinduced temporal and spatial changesin intestinal microbiota with associated alterations in immune cell homeostasismucosal immunol “ 101038mi2009132hooper l v xu j falk p g midtvedt t and gordon j i amolecular sensor that allows a gut commensal to control its nutrient foundationin a competitive ecosystem proc natl acad sci usa “ 101073pnas96179833ichinohe t pang i k kumamoto y peaper d r ho j h murray ts microbiota regulates immune defense against respiratorytract ‚uenza a virus infection proc natl acad sci usa “ 101073pnas1019378108inoue t nakayama j moriya k kawaratani h momoda r ito k gut dysbiosis associated with hepatitis c virus infection clin infectdis “ 101093cidciy205jiminez j a uwiera t c abbott d w uwiera r r and inglis gd impacts of resistant starch and wheat bran consumption onenteric ‚ammation in relation to colonic bacterial community structuresand shortchain fatty acid concentrations in mice gut pathog 101186s1309901601496karst s m the ‚uence of commensal bacteria on infection with entericviruses nat rev microbiol 101038nrmicro201525kauppila j h karttunen t j saarnio j nyberg p salo t gravesd e short dna sequences and bacterial dna induceesophageal gastric and colorectal cancer cell invasion apmis “ 101111apm12016kefalakes h and rehermann b ‚ammation drives an alteredphenotype of mucosalassociated invariant t cells in chronic hepatitis d virusinfection j hepatol “ 101016jjhep201905024khanna s and tosh p k a clinician™s primer on the role of themicrobiome in human health and disease mayo clin proc “ 101016jmayocp201310011kreuzer s machnowska p aŸmus j sieber m pieper r schmidt m f feeding of the probiotic bacterium enterococcus faecium ncimb diï¬erentially aï¬ects shedding of enteric viruses in pigs vet res krieg a m therapeutic potential of tolllike receptor activation natrev drug discov “ 101038nrd2059lee h and ko g antiviral eï¬ect of vitamin a on norovirus infection viamodulation of the gut microbiome sci rep 101038srep25835lemon s m ott j j van damme p and shouval d typea viral hepatitis a summary and update on the molecular virologyepidemiology pathogenesis and preventionj hepatol “ 101016jjhep201708034li d yan p abousamra a b chung r and butt a proton pumpinhibitors are associated with accelerated development of cirrhosis hepaticdecompensation and hepatocellular carcinoma in noncirrhotic patients withchronic hepatitis c infection results from erchives aliment pharmacolther “ 101111apt14391li h gao z zhang j ye x xu a ye j sodium butyratestimulates expression of fibroblast growth factor in liver by inhibition ofhistone deacetylase diabetes “ 102337db110846lin l and zhang j role of intestinal microbiota and metabolitesand human diseases bmc immunolon gut homeostasis 101186s1286501601873liu q li f zhuang y xu j wang j mao x alterationin gut microbiota associated with hepatitis b and nonhepatitis virus relatedhepatocellular carcinoma gut pathog 101186s1309901802816lu h wu z xu w yang j chen y and li l intestinal microbiotawas assessed in cirrhotic patients with hepatitis b virus infection microb ecol “ 101007s0024801098018meiglani a alimirah m ramesh m and salgia r fecal microbiotatransplantation for clostriodioides difficile infection in patients with chronicliver disease int j hepatol mencin a kluwe j and schwabe r f tolllike receptors as targets inchronic liver diseases gut “ 101136gut2008156307minemura m and shimizu y gut microbiota and liver diseases worldj gastroenterol 103748wjgv21i61691mohamadkhani aintestinal microbialcommunity in hepatocarcinogenesis in chronic hepatitis b cancer med “ 101002cam41550 on the potential role ofmoratalla a gmezhurtado i santacruz a moya ¡ peir g zapater p protective eï¬ect of bifidobacterium pseudocatenulatum cect against induced bacterial antigen translocation in experimental cirrhosisliver int “ 101111liv12380o™hara a mand shanahan ftherapeutic potential clin gastroenterol hepatolfor 101016jcgh200612009 gut microbiota mining“park e j thomson a b and clandinin m t protection of intestinaloccludin tight junction protein by dietary gangliosides in lipopolysaccharideinduced acute ‚ammation j pediatr gastroenterol nutr “ 101097mpg0b013e3181ae2ba0p©rezmatute p ­±iguez m villanuevamill¡n m j reciofern¡ndez e andv¡zquez a m s¡nchez s c shortterm eï¬ects of directactingantiviral agents on ‚ammation and gut microbiota in hepatitis cinfectedpatients eur j inter med “ 101016jejim201906005ponziani f r putignani l paroni sterbini f petito v picca a del chiericof ‚uence of hepatitis c virus eradication with directactingantivirals on the gut microbiota in patients with cirrhosis aliment pharmacolther “ 101111apt15004preveden t scarpellini e mili´c n luzza f and abenavoli l gut microbiota changes and chronic hepatitis c virus infection expert revgastroenterol hepatol “ frontiers in cellular and infection microbiology wwwfrontiersinaugust volume 0csehgal microbiota and liver diseasesraimondi s amaretti a gozzoli c simone m righini l candelieref in the human gutits profiling phenotyping and colonization front microbiol 103389fmicb201901575 longitudinalsurvey offungiren z li a jiang j zhou l yu z lu h gut microbiomeanalysis as a tooltowards targeted noninvasive bioma

" fasassociated factor faf1 has been implicated in parkinson™s disease pd and activates the celldeath machinery in the cytosol however the presence of extracellular faf1 has not been studiedmethods serumfree conditioned medium cm from faf1transfected shsy5y cells was concentrated andanalyzed by western blotting exosomes were isolated from cm by ultracentrifugation and analyzed by westernblotting electron microscopy and nanop tracking analysis soluble faf1 from cm was immunodepleted usingantifaf1 antibody transmission of secreted faf1 was examined by transwell assay under a confocal microscopecminduced cell death was determined by measuring propidium iodide pi uptake using a flow cytometerresults faf1 was secreted from shsy5y cells via exocytosis and brefeldin a bfaresistant secretory pathwaysfurthermore faf1 was secreted as a vesiclefree form and a genuine exosome cargo in the lumen of exosomes inaddition faf1 increased the number of exosomes suggesting a regulatory role in exosome biogenesis extracellularfaf1 was transmitted via endocytosis to neighboring cells where it induced cell death through apoptotic andnecrotic pathwayss this study presents a novel route by which faf1 induces neuronal death through celltocelltransmissionkeywords faf1 secretion exosome vesiclefree form celltocell transmission cell death cells secrete proteins harboring signal peptides throughthe classical secretory pathway via the endoplasmicreticulum ergolgi complex from which vesicles withcargo proteins move toward and fuse with the plasmamembrane and subsequently export cargos to the extracellular space however proteins lacking signal peptides are secreted via alternative nonclassical secretorypathways these pathways are classified as vesicularand nonvesicular secretory pathways some proteins correspondence eunheecnuackr gyeongrin park and bokseok kim are considered cofirst authorsdepartment of biological sciences chungnam national university daehakro yuseonggu daejeon south koreaare secreted via extracellular vesicles including exosomesand other vesicles of various sizes [ ] alternativelyother proteins are secreted through membrane poresand atpbinding cassette abc transporters althoughthe exact mechanisms of nonvesicular secretory pathways are elusive [ ]exosomes which are nanosized membrane vesicles “ nm in diameter secreted into the extracellular environment by various cell types are associated with intercellular communication with neighboring cells and play arole in a myriad of pathological functions in diseases including cancer cardiovascular and neurodegenerative diseasestheextracellular matrix and promote metastasis angiogenesisin particular exosomes[“]remodel the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpark cell communication and signaling page of thrombosis and tumor cell proliferation in cancer exosomes in cardiovascular disease display proangiogenesis procoagulant and proinflammatory effects on thevessel walls in neurodegenerative diseases exosomesare potential sources of key pathogenic proteins such astau amyloid prion and αsynuclein [“]inflammation triggersin addition to their vesiclemediated secretion manyproteins are secreted through nonvesicular mechanismsfor instance the proteins such as tau and fibroblastgrowth factor are recruited to the plasma membrane toform lipidic pores and are subsequently secreted [ ]in additionthe secretion ofinterleukin1 and transglutaminase through pores fibroblast growth factor translocates across the plasmamembrane via poorly understood mechanisms includingthe membrane release complex upon stress proteinssuch as hydrophilic acylated surface protein b are secretedby abc transporters proteins secreted via nonvesicular secretory pathways are advantageous over cargo proteins in vesicles as immunotherapeutic targets because ofthe antibody accessibility of the extracellular spacefasassociated factor faf1 is involved in diverse biochemical processes including cell death inflammation cellproliferation and proteostasis [“] consistent with itsdiverse functions faf1 has been implicated in certain diseases first faf1 plays a tumor suppressive rolethrough activation of the apoptotic machinery and nfκbsuppression [ ] faf1 also suppresses tumor metastasis via tgf signaling moreover faf1 expressionis downregulated in various cancers including lung colonliver prostate brain ovarian and breast cancers second faf1 is involved in parkinson™s disease pd as it iscolocalizes with αoverexpressed in pd patientssynuclein and acts as a substrate of parkin furthermorefaf1 mediates the degeneration of dopaminergic neuronsthrough apoptosis and parthanatos [“]faf1 is an intracellular protein present mainly in thecytosol to date faf1 secretion has not yet been reportedherein we uncovered for the first time that faf1 is secreted via an ergolgiindependent pathway specificallyfaf1 is secreted through exosomal and nonvesicular pathways in shsy5y cells in addition faf1 augments thenumber of exosomes suggesting the involvement of faf1in exosome biogenesis furthermore extracellular faf1moves into neighboring cells via pinocytosis and clathrinmediated endocytosis transmitted faf1 induces cell deathvia apoptosis and necrosis collectivelythese resultspresent a novel measure by which faf1 propagates itsdeath function through celltocell transmissionmethodsreagents and antibodiesthe following reagents and antibodies used in this studywere purchased commercially tnfα from abfrontierseoul south korea zietdfmk caspase8 inhibitormouse antiha antibody and rabbit antigm130 antibody from abcam cambridge uk ²²dichlorofluorescin diacetate dcfhda hydrogen peroxide h2o21methyl4phenylpyridinium mpp propidium iodidepi polydlysine brefeldin a bfa gw4869 monensin cycloheximide chx necrostatin1 nec1 dpqproteinase k pk dynasore heparin heparinase iiimouse antiactin and mouse antiflag antibody fromsigmaaldrich saint louis mo usa ²6diamidino2phenylindole dapi horseradish peroxidase hrpconjugated antimouse antibody and hrpconjugatedantirabbit antibody from thermo fisher scientific incrockford il usa mouse antiflotillin1 from bd biosciences san jose ca usa bafilomycin a1 mouseantialix antibody mouse antigalactosidase antibody401a rabbit anticalregulin antibody mouse anticd63 antibody mouse antiparkin antibody and mouseantifaf1 antibody from santa cruz biotechnologydallas tx usa mouse antihsc70 antibody andmouse antihsp90 antibody from enzo life sciencesfarmingdale ny usa and zvadfmk zvad fromcalbiochem darmstadt germanycell culture and transfectionshsy5y mef hek293 raw2647 hela panc1mia paca2 and mcf7 cells were maintained in dulbecco™s modified eagle™s medium dmem welgenedaegu korea containing fetal bovine serum fbsatlas biologicals fort collins co usa and antibioticantimycotic gibco brl grand island neusa unless otherwise specified cells were transfectedwith the indicated plasmids using bio t manvillescientific inc manville nj usa following the manufacturer™s protocol rat midbrain cultures derived frompostnatal day were prepared using standard procedures briefly material dissected form the ventral portion of the midbrain was cleaned free of meningealtissue minced and enzymatically dissociated in a mixture of papaindnase sigmaaldrich dissociated cellswere plated onto aminecoated 6well plates bd biosciences cells were maintained in neurobasal mediumgibco with b27 serumfree supplements gibco mm lglutamine after days of culture the cells wereinfected using aav1 adenoassociated virus 1hfaf1viral vectors moi of and sitedirected mutagenesisthe sirnaresistant parkin construct was generatedusing quikchange sitedirected mutagenesis kit stratagene la jolla ca usa the primers were as follows²gagctgagaaacgactggactgtgcagaattgtg3²²gggaaggagctgagaaacgattgcactgtgcaga ² 0cpark cell communication and signaling page of rna interferenceall small interfering rnas sirnas against parkin andscrambled rna scrna were purchased from bioneerdaejeon south korea the sequences of the sirnasused in this study were as follows sirna against parkin²ugaggaaugggacugu3² thescrna orsirna were transfected into shsy5y cells using lipofectamine rnai max thermo fisher scientific according to the manufacturer™s instructionspreparation of conditioned mediumto prepare conditioned medium cm cells transfectedwith the indicated plasmids were cultured in mmdiameter dishes in dmem containing fbs after h the cells were switched to serumfree dmem forthe indicated times here we used serumfree mediumto avoid interference from albuminenriched fbs thenthe cm was collected and centrifuged at ¨¯g for min and ¨¯g for min to remove cellular debrisfor western blot analysis the cm was concentratedusing kda or kda cutoff amicon ultra filtersmillipore billerica ma usa at ¨¯g for min or minwestern blot analysiscells were harvested washed twice with pbs and lysedwith mammalian lysis buffer [ mm triscl ph mm nacl mm edta nonidet p40 mmphenylmethylsulfonylfluoride] then the protein concentrations were quantified by using a biorad proteinassay kit biorad hercules ca usa after quantification samples were boiled in × protein sample buffer[ mm triscl ph glycerol sds mercaptoethanol] then samples were electrophoresedby sdspage and transferred to nitrocellulose membranes ge healthcare maidstone uk the membranes were blocked with skim milk in pbs with tween20 pbst and incubated with the indicatedprimary antibodies overnight after their washing withpbst the membranes were incubated with secondaryantibodies immunoblot signals were measured by usingchemiluminescent detection lab frontier anyangkoreachx chase assayshsy5y cells were transiently transfected with the indicated plasmids for h after transfection the cells wereswitched to serumfree dmem containing chx sigmaaldrich μgml for the indicated of times subsequently the medium was concentrated with kda cutoff amicon ultra filters millipore and analyzed bywestern blot analysispurification of exosomeswe followed a previously described protocol with somemodifications briefly cells were transfected withthe indicated plasmids for h and incubated in dmemcontaining exosomedepleted fbs system biosciences palo alto ca usa for h the cm was thencollected and subjected to sequential centrifugation at¨¯g for min ¨¯g for min and ¨¯g for min at °c to remove cellular debris using a beckmancoulter optima l90 k ultracentrifuge with a type 41tirotor the supernatant was then spun down at ¨¯gfor min the pellet was resuspended in pbs and thenspun again at ¨¯g for min at °c finally thepellet was resuspended in pbs or radioimmunoprecipitation assay ripa buffer sigmaaldrichin addition to their isolation via ultracentrifugationwe isolated exosomes using exoquicktc system biosciences according to the manufacturer™s instructionstreatment of vesicles with na2co3to separate integral membrane proteins and luminalproteins purified exosomes were treated with mmna2co3 ph for min at °c as previously described after centrifugation at ¨¯g for minintegral proteins remained in the pellet fraction whileluminal proteins remained in the supernatant fractionthe pellet fractions were resuspended in ripa buffersigmaaldrich and the supernatants were collected ina separate tube for western blot analysisproteinase k digestionpk sigmaaldrich was added to the samples at a finalconcentration of μgml then the samples were incubated at °c for min and mm phenylmethylsulfonyl fluoride was added to inhibit the activation of pkfollowed by the addition of protein sample bufferimmunodepletion of cmfifty microliters of protein gconjugated dynabeadsthermofisher scientific was incubated overnight withmouse monoclonal antibody against faf1 final concentration of or μgml before its addition to cm theantibodydynabeads complex was incubated with mlof cm at °c overnight after the complex was removedusing a magnet the immunodepleted cm was concentrated using a kda cutoff amicon ultra filter andused for western blot analysis for flow cytometryunconcentrated immunodepleted cm was applied to recipient cellsflow cytometryto evaluate cell death we measured pipositive cellstaining by using a guava easycyte flow cytometermillipore briefly cells were switched to serumfree 0cpark cell communication and signaling page of dmem or neurobasal medium for the indicated timeadditionally to measure recipient cell death shsy5yand rat primary neuronal cells were treated with cmfrom donor cells for the indicated times the cells were μgml and evaluatedharvested stained with piusing a guava easycyte flow cytometer following whichthe results were quantified using incyte softwaremilliporeconcentrated cm treatmentshsy5y cells donor cells were plated on mm tissueculture dishes and transfected with gfpvector or gfpfaf1 plasmid at h after transfection the cells wereincubated in serumfree medium for h after the cmwas concentrated by using kda cutoff amicon ultrafiltersthe concentrated cm was dissolved in newserumfree medium that was applied to recipient cellson polyllysinecoated coverslips in 12well plates for hpropagation assayshsy5y cells donor cells were plated on mm tissueculture dishes donor cells were transfected with gfpvector or gfpfaf1 plasmids at h after transfectionthe cells were collected and replated in cell culture inserts polycarbonate membrane μm pore size corning kennebunk me usa at a density of ¨¯ cellsshsy5y cells recipient cells were plated at a densityof ¨¯ cells on polydlysinecoated coverslips in well plates after h the cultures were combined suchthat the donor cells were in the insert and separatedfrom recipient cells plated on a coverslipconfocal microscopycells were fixed with paraformaldehyde for minthe cisgolgi were stained with gm130 after the nucleiwere stained with dapi for min the coverslips weremounted onto microscope slides using fluorescencemounting medium dako carpinteria ca usa andanalyzed using a zeiss lsm laser scanning confocalmicroscope carl zeiss oberkochen germanyelectron microscopyfor transmission electron microscopy tem sampleswere prepared using the exosometemeasy kit containing a formvarcarboncoated em mesh gridwash buffer and em solution bio mountain viewca usa the pellets from the ml of cm vector orfaf1transfected cells obtained by ultracentrifugationwere resuspended in μl of pbs μl of which was applied to the grid all samples were prepared followingthe manufacturer™s instructions for immunoem thepellets were first fixed with μl of paraformaldehyde and glutaraldehyde sigmaaldrich overnightat °c then the fixed exosome solution was transferredto grids and subsequently treated with m glycinefor min to quench free aldehyde groups after blocking with pbs containing bsa for min the gridswere incubated for h with the indicated antibodies diluted in pbs containing bsa atroomtemperature after three washes with pbs containing bsa the grids were incubated for h with the secondary antibody antimouse igg conjugated to nmgoldroomtemperature three washes to eliminate secondary antibody were followed by incubation with em solution anda wash step samples were viewed under a talos f200xtransmission electron microscope fei hillsboro orusa operated at kv and images were capturedwith a ceta m pixel cmos camera feisigmaaldrichatpsnanop tracking analysisfollowing isolation by differential ultracentrifugation orexoquicktc system biosciences the exosome pelletswere resuspended in μl of pbs then μl of theexosome solution was diluted in pbs to a total volumeof ml the samples were analyzed by nanoptracking analysis using a nanosight ns300 malvernpanalytical ltd malvern uk equipped with a nmlaser to accurately identify the vesicles the detectionthreshold was set at the number of vesicles in eachsample represents the number of ps per ml ofmedium cells were counted using a muse count viability kit millipore on a muse cell analyzer milliporecaspase3 activity assayshsy5y cells were treated with cm from faf1transfected cells plus caspase8 inhibitor or tnfα abfrontier plus chx sigmaaldrich at the indicated concentrations for the indicated times then caspase3 activity was measured by using a caspase3 colorimetricassay kit biovision milpitas ca usa in accordancewith the manufacturer™s protocol the absorbance at nm was measured with the use of a victor microplate reader perkinelmer norwalk ct usasignalp41we investigated the presence of a signal peptide in faf1using signalp41 httpwwwcbsdtudkservicessignalp41 with secretogranin1 used as a positive controlas it contains a signal peptidestatistical analysesexperiments were independently carried out three timesn all the data are expressed as the mean ± standarddeviation sd statistical comparisons were performedusing student™s ttest or oneway analysis of varianceanova followed by tukey™s hsd post hoc analysis 0cpark cell communication and signaling page of using spss software statistics version ibm incchicago il usa statistical significance was established when the pvalue was lower than resultsfaf1 is secreted via nonclassical exocytosisaccording to the csf proteome resource faf1 is detected in the cerebrospinal fluid and plasma additionalfile this study aimed to determine whether faf1 isalso secreted at the cellular level and investigate faf1secretion in shsy5y human neuroblastoma cellsbecause faf1 is a deathpromoting protein sublethal experimental faf1 transfection conditions wereused to exclude cellular debris due to death additionalfile fig s1a and b the faf1 transfection conditionsin shsy5y cells under which faf1 secretion but notcell death occurs were determined by pi stainingfollowed by flow cytometry cells were transfected with3xflagfaf1 plasmid at a sublethal dose for h andsubsequently moved to serumfree medium faf1 wasdetected in the serumfree medium at h and accumulated henceforth indicating that faf1 is secreted in atimedependent manner fig 1a this finding suggeststhat faf1 is constitutively released from shsy5y cellsto exclude a tagging artifact another epitope taghemagglutinin ha was used hafaf1 was alsodetected in the cm conditioned medium in a dosedependent manner additional file fig s1c furthermore we examined the faf1 secretion with rat primaryneurons using aav1 adenoassociated virus 1mediated faf1 overexpression faf1 overexpression in ratprimary neuronal cells demonstrated consistent resultsin shsy5y cells fig 1b moreover with thatgalactosidase was not detected in the cm of galactosidasetransfected cells demonstrating that therelease of faf1 is not a transfection artifact fig 1c next a pulsechase experiment using chx to inhibit de novo protein synthesis was performed consistently faf1 was secreted and accumulated over timefurther confirming that faf1 is constitutively secretedfrom shsy5y cells fig 1d next we examinedwhether faf1 is also secreted by other cells faf1 wasdetected in the extracellular space of mefs and hek293cells furthermore faf1 was present in the cm of eachof a number of various cancer cell types mcf7 helapanc1 and mia paca2 cells this shows that faf1secretion is notadditional file fig s2afspecific to shsy5y cellsbecause faf1 has been implicated in pd pathogenesisfaf1transfected shsy5y cells were treated with thepdassociated stressors such as mpp h2o2 bafilomycin a1 and αsynuclein overexpression at sublethal dosesadditional file fig s3 there were no significantchanges of faf1 expression in clslysatescelldependent on various pdassociated stressortypeshowever faf1 secretion increased in cms upon allstressors used in this study implying that pdassociatedstressors positively regulate faf1 secretion fig 1eto elucidate the mechanism by which faf1 is secreted two sets of experiments were performed as follows first we examined whether faf1 is released viaexocytosis or passive diffusion as exocytosis is affectedby temperature faf1transfected shsy5y cellswere incubated at either °c or °c faf1 secretionat °c was significantly reduced compared to that at °cindicating that faf1 is released via exocytosisfig 1f second we examined whether faf1 is secretedvia the classical ergolgidependent secretory pathwayusing bfa which generates ros and disassembles golgithrough ergolgi pathway inhibition bfa did not affectfaf1 release fig 1g additional file fig s1d furthermore a signal peptide was not found in faf1when its sequence was analyzed using singalp41 furtherexcluding the possibility of ergolgimediated secretionof faf1 additionalfile fig s4 taken togetherthese data demonstrate that faf1 is released via nonclassical exocytosisfaf1 is secreted via exosomal and nonvesicular pathwaysto determine the mechanism by which faf1 is secreted exosomes were isolated from cm using a differential ultracentrifugation procedure as previouslydescribed and exoquicktc following the manufacturer™s protocol western blot analysis of exosomesisolated by ultracentrifugation revealed the presenceofthe exosome markers alix cd63 hsc70 andhsp90 but not calregulin an er resident protein a negative exosome control fig 2a exosomes isolated from shsy5y cells by exoquicktc showed anexosome marker profile consistent with that of exosomes purified by ultracentrifugation additional file fig s5a moreover nanop tracking analysisand tem data further confirmed that our purifiedexosomes exhibited a typical size distribution with adiameter ranging from to nm and a typicalmorphology of exosomes fig 2b and cendogenous as well as overexpressed faf1 proteinswere detected in the exosomal fractions isolated by bothmethods indicating that faf1 is secreted as a genuineexosomal cargo fig 2a similarly faf1 was also foundin exosomes isolated from the various indicated cell linesby exoquicktc additional file fig s5bh next weinvestigated whether faf1 is present on the membraneor in the lumen of exosomes exosomes were treatedwith mm na2co3 ph to distinguish betweenthe exosomal membrane and the lumen both alix andflotillin1 were present in the exosomal membrane positive controls whereas faf1 was mainly present in the 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is secreted via nonclassical exocytosis a shsy5y cells were transfected with vector control vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium for the indicated times se short exposure le long exposure b ratprimary neuronal cells were transduced with aav1hfaf1 viral vectors at days after transduction the culture medium was replaced withserumfree neurobasal medium for h c cells were transfected with lacz or 3xflagfaf1 plasmid at h after transfection the culture mediumwas replaced with serumfree medium and cells were cultured for h d cells were transfected with vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium containing chx μgml for the indicated times e left panel cellswere transfected with vc or 3xflagfaf1 plus αsyn plasmid at h after transfection the culture medium was replaced with serumfreemedium containing dmso vehicle mpp mm h2o2 μm or baf a1 nm for h right panel the graph shows the densitometricanalysis of immunoblotting of faf1 in conditioned medium cm shown in the left panel n f upper panel cells were transfected with3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium and the cells were cultured at °cor °c for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting in cm shown in the upper paneln g upper panel cells were transfected with 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium containing bfa μgml for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting incm shown in the upper panel n cell lysate cl and concentrated cm were analyzed by western blotting with the indicated antibodies alllanes were loaded with the same amount of total protein the data are expressed as the mean ± sd of three independent experimentsstatistical comparisons were performed using using anova followed by tukey™s hsd post hoc analysis e and student™s ttest f and g p p p and ns not significanttheexosomeabolishedstructureslumen of the exosomes fig 2d the topology of exosomal faf1 was further examined using pk a nonspecificprotease to digest proteins outside of the exosomesexosomal faf1 but not cytosolic faf1 was protectedfrom pk treatment in the absence of triton x100 txfig 2e ˆ’tx in contrast pk treatment with tx disintegratingtheexosomemediated protection of faf1 fig 2e txour data demonstrated the presence of faf1 in thelumen of exosomes furthermore the presence of faf1in theconfirmed with theimmunogoldlabeled faf1 under anvisualization ofelectron microscope as shown in fig 2f immunogoldlabeled cd63 was mainly present outside exosomeswhereas immunogoldlabeled faf1 was present in thelumen of exosomes collectively these results consistently showed that faf1 is located in the lumen ofexosomeslumen wasexosomalbecause certain exosomal cargo proteins are alsosecreted via the nonvesicular secretory pathway [“] the possibility that faf1 is also secreted viaa nonvesicular route was investigated to this endthe cm was fractionated into exosomal pellet andnonexosomal supernatant fractions by ultracentrifugation faf1 from the cm was present in nonexosomal as well as exosomalfractions fig 2gimplying the presence of a soluble form of faf1 tofurther investigate this cm from faf1transfectedshsy5y cells was treated with antifaf1 antibodyone microgram of antifaf1 antibody almost eliminated faf1 from the cmindicating that faf1 ispredominantly secreted in a soluble form fig 2htaken together these results demonstrate that faf1is concurrently released as a bona fide cargo of exosomes and in a soluble form this new finding addsfaf1 to the list of proteins secreted by nonvesicularas well as exosomal routesrespectivelytransfection offaf1 positively regulates exosome numberthe exosomal cargos such as hsp20 hsp90 andstat3 increase exosome number [“] here weexamined whether faf1 also participates in regulating exosome number the exosome markers hsc70alix and cd63 were increased by ± 009fold ± 013fold and ± 022foldinthe cm of faf1transfected shsy5y cells compared with control cells fig 3a additional file fig s6a next we further studied exosome numberchanges using sirnamediated depletion of parkina e3ubiquitin ligase of faf1 siparkin treatment elevated secretion as well as expression of faf1 inshsy5y cells moreoversirnaresistant parkin construct reverted the increased secretion and expression of faf1 by siparkin in shsy5y cells the expression levels of alix and cd63in the cm of shsy5y cells in which parkin hadbeen depleted were elevated by ± and ± 040fold respectively fig 3b collectivelythese data imply that faf1 positively controls exosome number for the direct quantification of exosome number nanop tracking analysis wasapplied the vesicle size distribution profile showinga diverse range of sizes showed that exosomes werepresent predominantly fig 3c the exosome numbers were normalized by cell number additionalfile fig s6b the exosomes of faf1transfectedcells were increased by 25fold compared to thoseof control cells hence these data robustly demonstrate that faf1 augments exosome numberinaddition gw4869 an exosome release inhibitor interfered with the ability of faf1 to increase exosome number while monensin an exosome releasepromoter enhanced this ability implying that faf1functions upstream of the exosome release processfig 3a [ ] 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is released to the extracellular space via both exosomal and nonvesicular pathways a shsy5y cells plated on mm dishes weretransfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with exosomedepleted medium andthe cells were cultured for h then exosomes were isolated from the cm by ultracentrifugation cl and isolated exosomes exos wereanalyzed by western blotting with the indicated antibodies calr calregulin b purified exosomes were characterized using nanop trackinganalysis c representative tem images of exosomes are shown scale bar nm left or nm right d the purified exosomes were treatedwith na2co3 after centrifugation at ¨¯g the integral membrane proteins were pelleted memb and nonintegral and luminal proteinsremained in the supernatant sol these fractions were analyzed by western blotting with the indicated antibodies e the purified exosomeswere incubated with pk μgml in the absence or presence of triton x100 tx tx with tx ˆ’tx without tx f immunogold labelingof purified exosomes with anticd63 antibodies left and antifaf1 antibodies from vctransfected middle or 3xflagfaf1transfected rightcells scale bar nm g cells were transfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replacedwith serumfree medium and the cells were cultured for h after the cm was isolated by ultracentrifugation the supernatant sup and pelletexo were analyzed by western blot with the indicated antibodies h after immunoprecipitation of faf1 from cm with antifaf1 monoclonalantibody the immunodepleted cm was concentrated and analyzed by western blotting with the indicated antibodies all lanes were loaded withthe same amount of total proteinsecreted faf1 is transmitted to adjacent cellswe wondered whether extracellular faf1 is internalizedby neighboring cells donor cells were transfected withgfp or gfpfaf1 plasmid for h subsequently thecm which contained gfp or gfpfaf1 was concentrated and nontransfected recipient cells were treatedwith the concentrated cm for h confocal microscopic images showed the presence of gfpfaf1 in thecytoplasm of recipient cells indicating that gfpfaf1had moved from donor cells to recipient cells in contrast gfp from the cm of donor cells was not detectedin recipient cells excluding the effect of tagging ontransmission fig 4a similarly donor cells were transfected with 3xflagfaf1 plasmid as described abovedonor cellderived faf1 also moved into recipient cellsas shown by western blotting fig 4b corroborating theimmunofluorescence results in fig 4a in these data consistently demonstrate that secreted faf1can be internalized by neighboring cellsto further validate the celltocell transfer of faf1 anin vitro coculture system in which donor cells expressinggfpfaf1 were incubated in upper transwellinsertswhile nontransfected recipient cells were incubated inlower compartments was used consistent with theabove data confocal microscopy of recipient cells revealed the presence of gfpfaf1 indicating that gfpfaf1 moved from cells in the upper transwell inserts tocells in the lower compartments fig 4c consequentlythese results show that extracellular faf1 was transferred to neighboring cells without celltocell contactwe investigated the type of extracellular faf1 thatmoves into recipient cells to this end donor cells weretreated with gw4869 an exosome release inhibitorafter which recipient cells were analyzed by confocal microscopy gw4869 failed to inhibit faf1 transmissionto recipient cells to eliminate vesiclefree faf1 cm ofdonor cells was immunodepleted wit

"characterize the frequency and clinical features of lungnodules in IgG4 related disease IgG4RD patients as an insight for help with the diagnosis of lung nodulesMethods A retrospective study was carried out in West China Hospital Sichuan University from January toDecember patients with definite IgG4RD were enrolledResults Fifty of patients with definite IgG4RD had radiologically confirmed lung nodules of whom werediagnosed with definite IgG4 related lung disease Lung nodules detected in more than patients were small andsolid always with regular margins Multiple and bilateral distributions was also a major characteristicof these lung nodules Lobulation and speculation were simultaneously detected in patients including patientscombined with pleural indentation Calcification of nodules was detected in only one patient Thirtyseven patientsalso had additional radiological abnormalities of lungs including groundglass opacity thickening of pleura thickening of interlobular septa thickening of bronchial wall pleural effusion mass interstitial changes and mediastinal or hilar lymphadenopathy Most patients were treatedwith glucocorticoids alone or combined with immunosuppressive agents Sixteen patients received a reexamination by chest computed tomography CT scan after treatment of whom showed a decrease in the sizeandor the number of nodulesConclusions The incidence of lung nodules in IgG4RD patients can be high For an IgG4RD patient with lungnodules the possibility that the lung nodules related to IgG4RLD is high It is hard to differentiate IgG4 relatedlung nodules from other lung diseases in particular lung cancer Radiological characteristics and positive responsesto glucocorticoids and immunosuppressive agents can help with the differential diagnosis For these patientsregular followup is also importantKeywords Clinical characteristics IgG4 related disease IgG4 related lung disease Lung nodules RadiologicalcharacteristicsBackgroundClinical presentation of nodules in the lungs of patientscan be a challenge for diagnosis especially when thenodules are small and asymptomatic The nodules maybe difficult to biopsy for histopathology because of size Correspondence liuyihuaxiyiyuan126com Yan Xie and Anji Xiong contributed equally to this work1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu ChinaFull list of author information is available at the end of the and location for example very small nodules deeply imbedded in the lung The first diagnosis willlikely besome form of neoplasm When that diagnosis is determined to be incorrect many physicians especially respiratory physicians and thoracic surgeons may beunsure of the next step toward diagnosis and treatmentAffected patients of course will be anxious for a confirmed diagnosis since they will likely assume the smallnodules are an early stage of lung cancer A recently recognized diseaseimmunoglobulin G4related disease The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Pulmonary Medicine Page of IgG4RD must be considered for such presentation ofasymptomatic small nodules particularly in the lungsliver or lymph nodes []IgG4RD is a chronic fibrotic inflammation characterized by the involvement of multiple ans The mostcommon manifestations of the disease include swellingof salivary and lacrimal glands lymphadenopathy andtype autoimmune pancreatitis AIP Other anssuch as lung bronchi kidney retroperitoneum thyroidheart mesenterium meninges and skin can also be involved Frequently but not always serum IgG4 levelswill be elevated [] IgG4 is the rarest of the IgG subclasses generally has relatively low antigen affinity andis unable to bind complement component 1q C1q andactivate the complement cascade [] IgG4 can exchangeFab arms by swapping a heavy chain and attached lightchain with a heavylight chain pair from another IgG4molecule which providesthe antiinflammatory activity attributed to IgG4 antibodies [] Akey pathological feature of IgG4RD nodules is a denselymphoplasmacytic infiltrate anized into a storiformpattern which frequently forms a tumefactive mass thatmay destroy an involved an [] When the tumefactive mass occurs in the lungs it may present as a noduleor groundglass opacity on radiology [] IgG4relatedlung disease IgG4RLD is the lung involvement ofIgG4RD which was first described in in a patientwith interstitial pneumonia autoimmune pancreatitisand IgG4positive plasma cells in the interstitium []The IgG4RLD presentation can be heterogeneous andits radiologic manifestations are often extensive evenwhen clinically asymptomatic []the basisforAlthough lung nodules have been described in to of IgG4RLD in a few case reports and reviews todate [ ] no study has employed a systematic analysis of IgG4RD patients with lung nodules Here wepresent retrospective data of the clinical and radiologicalfeatures of IgG4RD patients from a single medical center™s experience Our goal is to provide clinical information and insight for the diagnosis of IgG4RD in patientswho present with asymptomatic nonneoplastic smalllung nodulesMethodsPatients informationPatients with definitive IgG4RD were selected from alldepartments of West China Hospital Sichuan Universityfrom January to December The clinical serological and radiographic imaging characteristics andtreatment responses of the patients were analyzedOur study was performed according to the rules of thehospital™s medical ethics committee Informed consentwas obtained in accordance with the institutionalguidelinesDiagnosis of IgG4RDThe diagnosis of IgG4RD was made according to thecomprehensive diagnostic criteria for IgG4RD publishedin by Umdehara [] The criteria for definiteprobable and possible IgG4RD are as followsDefinite1 Probable1 Possible1 Diffuse or localized swelling or masses in single ormultiple ans Elevated serum IgG4 mgdl Histopathology of biopsied nodules showsa Marked lymphocyte and plasmacyte infiltrationand fibrosisb Infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cells and IgG4 plasma cells per 40X fieldChest CT protocolsCT images were available for all patients involved in thisstudy Because of the retrospective nature of this studychest CT scanning protocols were varied The scanningwas performed on one of the six machines ranging from16detector to 128detector CT scanners Philips Medical Systems Best the Netherlands or Siemens MedicalSystems Erlangen Germany with patients in the supineposition For patients who underwent contrastenhancedexamination an intravenous nonionic contrast mediumIopamidol mgml ml was given and imaging started s later after injection To minimize motion artifacts CT images were acquired during a singlebreathhold The main parameters were shown as follows tube voltage 100120kv tube current mA slice thickness mm section interval mmDefinition of lung nodules and IgG4RLDCT images of patients™ chests were analyzed by radiologists The lung nodules were defined as rounded or irregular opacity well or poorly defined measuring up to cm in diameter [] Nodules were categorized aslarge or small as follows large cm ‰ diameter ‰ cmor small diameter cm If the diameter of the lung lesion is larger than cm in the CT images the lesionwas defined as a massIgG4RLD is defined as IgG4RD diagnosed as abovethat includes diffuse or localized swelling or masses inone or both lobes of the lungs with histopathology thatincludes marked lymphocyte and plasmacyte infiltrationand fibrosis andor infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cellsand IgG4 plasma cells per × field 0cXie BMC Pulmonary Medicine Page of StatisticsStatistical analyses were performed with GraphPadPrism version GraphPad Software Inc La Jolla CAUSA Descriptive data are reported as median interquartile range and frequencies are percentages Continuous variables without normal distribution wereexpressed as median and interquartile IQR For comparison between groups the unpaired Student™s ttestwas used for continuous variables and chisquare testwas used for categorical variables All statistical testswere 2sided and results with P values were considered statistically significantResultsFrom January to December a total of patients were diagnosed with definitive IgG4RD in WestChina Hospital Sichuan University of whomhad CTconfirmed lung nodules were included in thisretrospective studyPatient description and clinical presentationDemographic and clinical data for all patients are summarized in Table The median age of the patients withlung nodules was “ years at diagnosis with amale female ratio of Almost half of the patientshad their initial diagnosis of IgG4RD in the Departmentof Rheumatology The duration of symptoms beforediagnosis averaged months range “ monthsFortytwo of the patients had no pulmonary infectionor a history of cancer and other chronic pulmonary diseases And more than half of the patients had ahistory of smokingFirst symptomsFor patients with lung nodules the most common firstsymptoms were salivary gland swelling and lacrimal gland swelling Seven patients presented withcough as their first symptom There were several raresymptoms including fever gum swelling low back painand edemaExtrapulmonary ans involvedMost patients had multiple sites or ans involved median number IQR1“ Twentyone patients had atleast three extrapulmonary ans or sites affected Themost common involved extrapulmonary ans includedsalivary gland lacrimalgland pancreas and kidney Other sites included liver nasal sinus peritoneum pituitaryskinthyroid glands bile ducts gingiva andpericardiumlymph node Serological characteristicsSerum levels of IgG4 erythrocyte sedimentation rateESR Creaction protein CRP and complements werequantified Serum IgG4 concentrations were measuredin all patients in this study with a median value of mgdl IQR426“ for patients with lung nodulescompared to mgdl IQR371“ for IgG4RD patients without lung nodules Table Elevated ESR andserum CRP levels were found in and patients respectively among patients with lung nodules versus patients with elevated ESR and patients with elevatedCRP among IgG4RD patients without lung nodulesTwentyfour patients with lung nodules versus without had reduced serum complement Twenty patientswith lung nodules versus without had antinuclearantibody ANA was defined as positive Therewere no statistically significant differences in serologicalmeasurements between IgG4RD patients with and without lung nodules Table Comparison of clinical characteristicsThe general clinical characteristics of patients with lungnodules were compared with those of patients withoutlung nodules As the results indicated in Table significant differences were observed in smoking history between the two groups while no significant differences inage gender ratio duration of symptoms before diagnosis and the number of extrapulmonary involved answere observedRadiological characteristics of the lungsFifty patients included in this study had lung nodules revealed in chest CT images The radiological characteristics of lungs among the patients with lung nodulesare summarized in Table and Table Five patients had large lung nodules Fig 1a with diameters ranging from to cm while almost all patients showed smalllung nodules Fig 1beMost patients had multiple nodules Only patients had single small nodules For the distribution oflung nodules patients had bilateral nodules accounting for more than half of the cases When consideringthe pulmonary lobe involvement the number of patientswith nodules located only in the upper lobe and middleor lower lobe was and respectively The rest patients had nodules randomly distributed in differentlobes at the same timeFor the lesion densities on CT solid nodules can bedetected in almost all patients Only patientsshowed groundglass nodules all of which were smallnodules Nodules in out of the patients showed bothgroundglass and solid patterns Fig 1d Fig 1e Mostpatients showed regular and welldefined nodulemargins Only patients had nodules with irregular or 0cXie BMC Pulmonary Medicine Page of Patients with lung nodulesn “Patients without lung nodulesn “““““Table Clinical characteristics of IgG4RD patientsParametersAge medianIQR yearsMenwomen n Smoking history n Duration of disease medianIQR monthsSerum IgG4 medianIQR mgdlElevated ESR n Elevated CRP n Reduced C3 n Reduced C4 n Positive ANA n First symptoms n Salivary gland swellingLymphadenopathyLacrimal gland swellingCoughJaundiceDysuriaFeverAbdominal painGum swellingLow back painEdemaFatigueDiarrheaNumber of extrapulmonary ans involved medianIQRExtrapulmonary an involvement n “Salivary glandssubmandibular gland and parotid glandLymph nodeLacrimal glandPancreasKidneyLiverNasal sinusSkinBile ductsPeritoneumPituitaryThyroid glandsGingivaPericardiumBloodP value“Data are expressed as medianinterqurtile range for contious variables and frequenciespercentages for categorical variablesIgG4RD IgG4 related disease ESR Erythrocyte sedimentation rate CRP Creactive protein C3 Complement C4 Complement ANA Antinuclear antibody IQRInterquartile range P 0cXie BMC Pulmonary Medicine Page of Table Radiological characteristics of lungs before drugtherapyPatients with indicated lung nodulesSizesinglemultipleSmall nodule onlyLarge nodule onlyBoth small and large noduleDistributionLateralityUnilateralBilateralLobeUpper lobeMiddle or lower lobeRandomTypeGroundglass nodule onlySolid nodule onlyBoth groundglass and solid noduleMarginIrregular or untidyRegularSecond associated featuresLobulationSpiculationPleural retractionCalcificationChanges of nodules after treatmentsn Smaller or disappearNo differenceOther radiological featuresGroundglass opacityThickening of pleuraThickening of interlobular septaThickening of bronchial wallPleura effusionMassConsolidationInterstitial changesn Mediastinal and hilar lymphadenopathypoorly defined margins Some other features of nodulesincluding lobulation spiculation pleuralindentationand calcification were also explored All these signswere rare in our study Lobulation and speculation Fig1c were simultaneously detected in patients including patients combined with pleural indentation Fig 1cCalcification of nodules was detected in only one patientThirtyseven patients including the patients with definite IgG4RLD showed some other radiological abnormality of lungs Fig which included groundglassopacity2150 thickening of pleura950 thickening ofinterlobular septa thickening of bronchial wall3 pleural effusion450 mass350 consolidation1 interstitial changes550 and mediastinal or hilarlymphadenopathy3250Six patients were diagnosed as definite IgG4RLD Thechest CT findings of these patients were shown in Table All of the patients showed mixed patterns in CTchanges Two patients had single large lung nodulesonly while the rest patients showed multiple smalllung nodules only Mass thickening of pleura thickening ofinterlobular septa groundglass opacity andpleura effusion were detected in and patientsrespectively Enlarged mediastinal or hilar lymph nodewas detected in all IgG4RLD patientsTherapy and responsesThe results of therapy and responses are shown in Table and Table For the treatment of the patients withlung nodules patients received prednisone with orwithout additional antiinflammatory or immunosuppressive drugs with prednisone only with prednisone and cyclophosphamide with prednisone andmycophenolate mofetil or methotrexate and withprednisone and azathioprine Three patients received asurgical treatment only and patients with mild diseasereceived symptomatic therapyMost patients improved to some degree aftertreatments during the duration of hospitalization Sixteen patients received a reexamination of chest CT scanafter treatment The durations between CT scans wereat least month Ten patients showed a decrease in thesize andor the number of nodules while patientsshowed no difference between pre and posttherapy CTimagesDiscussionLung nodules are small rounded lesions with at leasttwothirds ofits margins surrounded by lung parenchyma and not associated with atelectasis or lymphadenopathy In this study we focused on the lung nodules ofIgG4RD patients and IgG4RD patients presented with lung nodules in CT images As far as weknow there hasn™t been any study concerned about theincidence of lung nodules in IgG4RD patients Previousstudies have reported that lung nodules were incidentallyidentified in approximately “ ofthe socialdemographically population [ ] Our result revealed 0cXie BMC Pulmonary Medicine Page of Table Radiologic findings in patients with definite IgG4RLDPatientsMass Thickening ofNoGroundglassopacityYespleuraYesSmallnoduleLargenoduleSolitaryThickening ofinterlobular septaPleuraeffusionEnlarged mediastinal or hilarlymph nodeYesSolitaryYesYesMultipleMultipleMultipleMultipleYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesthat the incidence of lung nodules in IgG4RD patientswas much higherComparing the clinical characteristics of patients withlung nodules with that of patients without lung nodulesno significant difference was found in terms of age gender ratio duration of symptoms before diagnosis number of extrapulmonary involved ans and serologicalcharacteristics indicating that these factors have no association with the progress of nodule formation Forclinical symptoms only patients were observed withcough Most patients especially patients with small nodules only were relatively asymptomatic despite substantial burdens of disease within thelung Clinicalsymptoms of lung disease depend on the location andsize of lesions and are often nonspecific for the diagnosisof some lung disease including IgG4RLDIn our study six patients were diagnosed as definiteIgG4RLD All of the patients had lung nodules Consistent with results of previous studies conducted byInoue [] and Sun [] this result revealedthat nodular lesion can be a common manifestation ofIgG4RLD Together with lung nodules some otherchest CT findings including mass solid nodules roundshaped glass opacity thickening of bronchovascular bundles and interlobular septa alveolar interstitial changeslike honeycombing and bronchiectasis lobar or segmental consolidation and lymph node enlargement et alwere often seen in IgG4RLD patients And these CTchanges always present as various mixed patterns [ “] In this study except lung nodules we also detected mass groundglass opacity thickening of pleurapleura effusion mediastinal and hilar lymphadenopathyand thickening of interlobular septa in IgG4RLD patients It is worth noting that IgG4RLD related nodulelesion reported previously are always single solid andlarge type together with or without multiple small nodules [ “] And these nodules are always solidand with spiculated or irregular margins In our studyonly IgG4RLD patients had large nodules while ofthe IgG4RLD patients presented as multiple smallnodules And only one of the patients had noduleswith irregular margins As pulmonary biopsy results areFig Radiological manifestations of lung nodules in IgGRD patients via chest computed tomography scans a A single large solid nodule withirregular margin was detected in the right lung Thickening of pleura and tracheal traction were also noted b Multiple small and solid lungnodules were scattered in both lungs c A single small and solid nodule was shown in the right lung apex Lobulation spiculation and pleuralindentation of the nodule can be noted Thickening of pleura and the bronchial wall can also be observed de Multiple solid and groundglassnodules were shown in both lungs 0cXie BMC Pulmonary Medicine Page of Fig Other radiological manifestations of lungs in IgGRD patients via chest computed tomography scans a Thickening of the left pleura canbe observed A small nodule near the thickened pleura can also be noted bc Thickening of interlobular septa and pleura effusion in the rightlung can be observed d Mass and groundglass opacity were shown ef Small lung nodules combined with groundglass opacity consolidationthickening of interlobular septa and thickening of bronchial wall can be observedhard to get this inconsistency may be caused by a smallsample size of studies related to IgG4RLD Thus morestudies are needed to clarify the CT imaging features ofIgG4RLDAs lung nodules could also be caused by infection malignancy or some other pulmonary disorder we cannotbe certain that in the absence of definitive nodular biopsies all of the lung nodules in our study were related toIgG4RLD For an IgG4RD patient with lung nodulesthe probability of lung nodules related to IgG4RLD isvery high especially in the absence of other lung diseases In a study reported by Tsushima of IgG4RD patients with lung nodules were confirmed tobe IgG4RLD by biopsy [] In our study most patientshad no pulmonary infection or a history of cancer andother chronic pulmonary diseases For these patientsthe probability of lung nodules related to IgG4RLD isvery high Besides CT findings of our study also revealedthat most of the lung nodules in IgG4RD patients weresmall and solid always with regular margins MultipleTable Treatments of IgG4RD patientsTreatmentsPatients with lung nodulesn Pred onlyPredimmunosuppressiveagentsothersPredCYC PredMMF PredMTX PredAZA IgG4RD IgG4 related disease Pred prednisone CYC cyclophosphamide MMFmycophenolate mofetil MTX methotrexate AZA azathioprine Others surgicaltreatment or symptomatic no coticosteriod or immunosuppressive agentswas givenand bilateral distributions was also a major characteristicof these lung nodules Lung nodules in one patient alsoshowed calcification These radiologic features and distribution of lung nodules were usually regarded as benign [ ]IgG4RLD variedIt is noteworthy that three patients with large lunglobulation spiculation andnodules showed signs ofpleuralindentation all of which may predict an increased risk of malignancy [ ] As radiological findings ofthese signs can often beobserved in lung nodules and masses related to IgG4RLD [ ] Besides we also found that the proportionof lung nodules in smokers was significantly higher thanthat in nonsmokers indicating that smoking may be arisk factor of these lung nodules The proportion of elderly in IgG4RD patients was not small Therefore forIgG4RD patients with lung nodules it™s necessary buthard to differentiate IgG4RLD from lung cancer Asmentioned above the simultaneous presence of otherkinds of lung lesions may provide support for the diagnosis of IgG4RLD and help with the differential diagnosis In our study most patients also had thesimultaneous presence of other CT findings includingthickening of pleura thickening of interlobular septathickening of bronchial wall pleural effusion presenceof an undefined mass interstitial changes consolidationgroundglass opacity and mediastinal or hilar lymphadenopathy which may help to increase the possibilityof IgG4RLDMost IgG4RLD patients have a significant response toglucocorticoid therapy which can help to further distinguish IgG4RD nodules and malignancy [] In ourstudy patients went through a reexamination of CTscan after glucocorticoid therapy with or without 0cXie BMC Pulmonary Medicine Page of immunosuppressive agents Ten patients including patients that had large lung nodules with signs of lobulation spiculation pleural indentation and cavity showedpositive responses A decrease in the size andor thenumber of nodules was observed which helped to further support the diagnosis of IgG4RLDtype oflobectomy forSometimes during a routine medical examinationpatients may present with lung nodules detected without other pulmonary symptoms Since nodules mayresemble bronchoalveolar carcinoma and raise suspicion of malignancy [] case reports indicate that patients with thislung lesions may haveundergone wedge resection orsuspected malignancy only to discover upon tissue examination that the relevant nodules were a consequenceof IgG4RD [ ] In our study a patient with bilateral smalllung nodules was first misdiagnosed astuberculosis and another patient with a single largenodule underwent a middle lobectomy and histological findings were consistent with IgG4RD Thusan understanding of nodules related to IgG4RD isnoteworthy Since IgG4RD may be a multian disease the involvement of other ans and serologicalchange can help with diagnosis in patients with undiagnosed lung nodules In our study only patientshad the pathological proof of IgG4RLD while theother patients were diagnosed with IgG4RD primarilybecause of clinical manifestations in other ansparticularly salivary glandslacrimalgland and pancreas which is consistent with otherstudies [] In Sun™s study of biopsyproven IgG4RLD patientsinvolvement wasproven in only patient with uveitis mastoiditisSince patients with only lung involvement are moreprone to receive a lung biopsy the uncommon extrapulmonary involvement may be caused by selectionbias [] Once the diagnosis of IgGRD is confirmedthe radiological characteristic and treatment responsecan help with the diagnosis ofthus aregular followup is important for these patientsextrapulmonarylymph nodesIgG4RLDOur study had some limitations First most of the lungnodules involved in our study were not histopathologically proven Therefore we were unable to clarify theexact proportion of lung nodules related to IgG4RLDSecond due to the retrospective features of our studythe scan protocols of CT varied and the number of patients with a reexamination of CT was small whichmay further limit the value of our studyConclusionsThe incidence of lung nodules in IgG4RD patients canbe high For an IgG4RD patient with lung nodules thepossibility that the lung nodules related to IgG4RLD ishigh It is hard to differentiate IgG4 related lung nodulesin particularfrom other lung diseaseslung cancerRadiological characteristics and positive responses toglucocorticoids and immunosuppressive agents can helpwith the differential diagnosis For these patients regularfollowup is also importantAbbreviationsIgG4RD IgG4 related disease AIP Autoimmune pancreatitisC1q Component 1q IgG4RLD IgG4related lung disease CT Computertomographic IQR Interquartile range ESR Erythrocyte sedimentation rateCRP Creaction protein ANA Antinuclear antibody C3 Complement C4 Complement Pred Prednisone CYC CyclophosphamideMMF Mycophenolate mofetil MTX Methotrexate AZA AzathioprineAcknowledgementsNot applicableAuthors™ contributionsYX and AX equally contributed to study design data collection andinterpretation and drafting and revisions of the manuscript TM contributedto drafting and revision of the manuscript YL contributed to drafting andrevision of the manuscript All authors have read and approved themanuscriptFundingThis work was not funded by any grantsAvailability of data and materialsAll relevant data in this study are freely available to any scientist wishing touse them for noncommercial purposes without breaching participant confidentiality And relevant data can be obtained by contacting the corresponding authorEthics approval and consent to participateThe study was approved by the Ethical Committee of West China HospitalSichuan University According to the rules of the hospital™s medical ethicscommittee this study fulfilled the criteria of exception to the requirementsof informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu China 2Department of MicrobiologyImmunology and Biochemistry University of Tennessee Health ScienceCenter Memphis TN USAReceived August Accepted July ReferencesStone JH Zen Y Deshpande V IgG4related disease N Engl J Med “Vasaitis L IgG4related disease a relatively new concept for clinicians Eur JIntern Med “Doi T Kanatsu K Mayumi M Analisis of IgG immunecomplexes in serofrom patients with membranous nephoropathyrole of IgG4 subclass andlowavidity antibodies Nephron “van der Neut KM Schuurman J Losen M Bleeker WK MartínezMartínez PVermeulen E den 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management of incidental pulmonary nodules detected onct images from the fleischner society Radiology “ Masaki Y Kurose N Yamamote M Takahashi H Saeki T Azumi A Cutoffvalues of serum IgG4 and histopathological IgG41 plasma cells for diagnosis ofpatients with IgG4 related disease Int J Rheumatol “ Okubo T Oyamada Y Kawada M Kawarada Y Kitashiro S Okushiba SImmunoglobulinG4related disease presenting as a pulmonary nodule withan irregular margin Respirol Case Rep 201651e00208 Odaka M Mori S Asano H Yamashita M Kamiya N Morikawa TThoracoscopic resection for a pulmonary nodule with the infiltrate of IgG4positive plasma cells Asian J Endosc Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we briefly summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microfilament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was first discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as protein“protein interaction modules act as scaï¬olds to bindto filamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ˆ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identified one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially defined cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc fingerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin filamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespecificexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17ˆ¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microfilamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespecific scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the first transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspecific PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a significantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout significantlydecreases the expression of these two proteins these findingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a specific scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspecific antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha specific distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the specific molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialfibrosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac fibroblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17ˆ¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac fibroblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspecific knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17ˆ¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17ˆ¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above findings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identification of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microfilamentassociated proteinsMicrofilamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin filaments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Thesefindings show that the PDZLIM protein plays an overall role incell“cell and cell“matrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microfilamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress fibersand responds to stress by directly binding to the key stressfiber component αactinin Microfilaments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of significant amountsof a vinculinpositive adhesion complex on a hard substratecoated with fibronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspecific PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we briefly reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe specific molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicrofilamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl “ 101002prcaBach I The LIM domain regulation by association Mech Dev “ 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J “ fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer “ 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac fibroblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the specific mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identified Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modificationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest “ 101172JCI70577Barry S P and Townsend P A What causes a broken heart“molecularinsights into heart failure Int Rev Cell Mol Biol “ S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase nature™s energy sensor Nat Chem Biol “ nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17ˆ¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149“L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med “ 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen “ Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res “ 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modifiers in protein interactions Trends Genet “ 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature “ 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer “ 101002ijc27518Eeckhoute J A celltypespecific transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev “ 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl

Overexpressed EphB4 conduce to tumor development and is regarded as a potential anticancer target HomoharringtonineHHT has been approved for hematologic malignancies treatment but its effect on hepatocellular carcinoma HCC has notbeen studied This study elucidated HHT could restrain the proliferation and migration of HCC via an EphB4catenindependent manner We found that the antiproliferative activity of HHT in HCC cells and tumor xenograft was closelyrelated to EphB4 expression In HepG2 Hep3B and SMMC7721 cells EphB4 overexpression or EphrinB2 Fcstimulation augmented HHTinduced inhibitory effect on cell growth and migration ability and such effect wasabrogated when EphB4 was knocked down The similar growth inhibitory effect of HHT was observed in SMMC and EphB4SMMC7721 cells xenograft in vivo Preliminary mechanistic investigation indicated that HHTdirectly bound to EphB4 and suppressed its expression Data obtained from HCC patients revealed increasedcatenin expression and a positive correlation between EphB4 expression and catenin levels HHTinducedEphB4 suppression promoted the phosphorylation and loss of catenin which triggered regulation of catenindownstream signaling related to migration resulting in the reversion of EMT in TGFinduced HepG2 cellsCollectively this study provided a groundwork for HHT as an effective antitumor agent for HCC in an EphB4catenindependent mannerIntroductionGlobally hepatocellular carcinoma HCC is one of themost fatal malignancies with poor prognosis and anincreasing incidence1 Although the major therapeuticapproaches such as surgical resection radiation therapyand chemotherapy have advanced clinical applicationsthe 5year survival rate of HCC remains less than Most patients still suffer from tumor recur invasivenessand metastasis At present sorafenib a multiple tyrosinekinase inhibitor is one of the most representative optionsfor advanced HCC butlimited andaccompanied with reduced sensitivity and severe adversesometimesisCorrespondence Yanmin Zhang zhang2008mailxjtueducn1School of Pharmacy Health Science Center Xi™an Jiaotong University No Yanta Weststreet Xi™an Shaanxi PR ChinaEdited by B Zhivotovskyevents34 Therefore much effort is needed on this frontto uncover new antiHCC therapeutic strategies5Erythropoietinproducing hepatocytereceptor B4EphB4 is a member of the tyrosine kinase family andplays a pivotal role in tumor progression6“ Activatedby its corresponding ligand EphrinB2 EphB4 controlscell“cell interactions angiogenesis tumor growth andmetastasis910 Studies on the expression of EphB4 innumerous cancer types have shown overexpressed levelin breast colorectal lung and blood cancers correlatingwith poor prognosis11“ It has been reported that highEphB4 expression enhanced the growth and migrationof pancreatic colorectal and papillary thyroid carcinoma and such effect could be reversed by EphB4knockdown making EphB4 a promising target for cancer treatment14“ Our previous study has confirmed The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT exhibited a growth inhibitory effect on HCC cells in vitro and in vivo a The chemical structure of HHT b Effects of HHT on cellproliferation in Hep3B HepG2 SMMC7721 Bel7402 and Bel7404 cells were determined by MTT assay p comparedto the IC50 of HepG2 cells Cells were treated with increased gradients of HHT for h n cultures for each dose c Protein expression of EphB4 inHep3B HepG2 and cells d Quantification of c n independent experiments e Effects of HHT on colony formation in HepG2cells The upper row the colony formation picture the lower row the individual colony picture — magnification f Photographs of control andHHTtreated group tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor inhibitory rate n mice g h data represent mean ± SEM p p compared to vehicle controls i Inhibitory rate of HHT on tumor mass n micethe high expression of EphB4 in HCC17 and its functionin HCC migration remains poorly understoodHomoharringtonine HHT Fig 1a is a compoundextracted from traditional Chinese medicine and has beenapproved for the treatment of leukemia by Food and DrugAdministration18 Previous studies indicated that HHTcould suppress protein synthesis essentialfor cancersurvival and induce apoptosis by upregulating the proapoptotic protein Bax and inducing caspase3mediatedcleavage of PARP19 In addition to hematologic tumorsHHT also demonstrated its effectiveness in renal cellcarcinoma colon rectal cancer and nonsmall cell lungcancer20“ However the effect of HHT on HCC and theunderlying EphB4related mechanism of action have notbeen studied In this study HHT was found to suppressthe proliferation and migration of HCC cells through anEphB4catenin dependent mannerResultsHHT exhibited a growth inhibitory effect on HCC cellsin vitro and in vivoTo determine the effect of HHT on the cell viability ofHCC cells several different HCC cells HepG2 Bel7402Hep3B and SMMC7721 were treated with an increasedgradient of HHT for h The results showed that HepG2cells were most sensitive to HHT treatment with an IC50value of μM while the IC50 values of Bel7402Hep3B Bel7404 and SMMC7721 cells were and μM respectively Fig 1b Immunoblotting analysis showed that HepG2 cells exhibitedhigher EphB4 expression Fig 1c d suggesting thepositive correlation between the inhibitory effect of HHTand EphB4 expression Similar results were obtained fromthe colony formation assay HHT significantly reduced thecolony size and the number of HepG2 cells at a doseOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig The inhibitory effect of HHT on HCC cells was associated with EphB4 expression a EphB4 expression analysis of EphB4siRNA or EphB4overexpression OE HepG2 cells b Effects of HHT on cell proliferation in wildtype EphB4siRNA EphB4OE or EphrinB2 Fc stimulated HepG2 cellsn cultures for each dose p compared to the IC50 of HepG2 cells c EphB4 expression analysis of EphB4OE Hep3B cells d Effects of HHTon cell proliferation in wildtype and EphB4OE Hep3B cells n cultures for each dose p compared to the IC50 of Hep3B cells e EphB4expression analysis of wildtype and EphB47721 cells f Photographs of control and HHTtreated group of tumors and EphB47721tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor mass n mice i Body weight ofcontrol and HHTtreated group mice n g“i data represent mean ± SEM p compared to vehicle controlsdependent manner in comparison to the control groupFigs 1e and S1a Moreover xenografts model of HepG2cells confirmed the antitumor effect of HHT in vivo HHTgavage groups showed remarkable reduction in tumrowth Fig 1f“i and the inhibitory rate reached and at the mgkg mgkg and mgkg inHHT gavage groups respectivelyThe inhibitory effect of HHT on HCC cells was associatedwith EphB4 expressionTo evaluate whether the proliferation inhibitory effectof HHT on HCC cells was related to EphB4 expressionEphB4 siRNA or plasmid was utilized to transfect theHCC cells Figs 2a and S1b and EphrinB2 Fc was used tostimulate the HCC cells As is shown in Fig 2a b HepG2cells with EphB4 knockdown were less sensitive to HHTwhereas HepG2 cells with EphB4 overexpression EphB4OE demonstrated elevated sensitivity to HHT treatmentcompared with wild type HepG2 cells HepG2 cells following EphrinB2 Fc stimulation showed a drug responsecurve that was similar to that of EphB4 OE subline Fig2b Meanwhile following transfection with EphB4 plasmid Hep3B cells harboring high expression ofEphB4 showed less cell viability after HHT treatmentcompared with wild type Hep3B cells Fig 2c d Forin vivo test an EphB4overexpressing SMMC7721EphB47721 cell line was established Figs 2e and S1cand the antitumor effect of HHT on xenograft model ofOfficial journal of the Cell Death Differentiation Associationcellsand EphB4wild type SMMC7721 cells was investigated HHT has an enhanced inhibitory effect on EphB47721 tumor growth comparedwith that on wild type tumor Fig 2f“h And therewas no obvious body weight and spleen index reductionduring the test Figs 2i and S1dThe suppression of HHT on SMMC7721 cells migrationwas associated with EphB4Migration assay and wound healing assay were conducted to investigate the effect of HHT on HCC cellmigration The results showed that HHTtreated wide typeSMMC7721 cells had decreased migration as comparedwith controls whereas both of EphB4 overexpression andEphrinB2 Fc stimulation in SMMC7721 cells strikinglyenhanced migration restraint effect of HHT Fig 3a cSimilar result was observed in wound healing assay whichdemonstrated that both transfection with EphB4 andexogenous stimulation with soluble EphrinB2 Fc inSMMC7721 cells delayed the closure of wound gapsfollowing HHT treatment Fig 3b d These results indicated that the suppression of HHT on HCC cells migration was closely associated with EphB4 expressionHHT suppressed HepG2 cell migration induced by TGFstimulationTGF stimulation could induce EMT and increase themigration of tumor cells We next investigated the effect 0cZhu Cell Death and Disease Page of Fig The suppression of HHT on SMMC7721 cell migration was associated with EphB4 a Transwell assays were conducted to observe themigratory cells in HHTtreated wide type EphB4OE or EphrinB2 Fc stimulated cells Scale bars μm b The migration rate of HHTtreated EphB4OE or EphrinB2 Fc stimulated cells observed through woundhealing assays Scale bars μm c Quantification of a n Leftp compared to the migrated cell number of cells Right p and p compared to the migration rate of cells atindicated concentration of HHT d Quantification of b n p compared to HHTtreated cells All data represent mean ± SEMof HHT on HCC cells migration after TGF stimulationby transwell migration assay and wound healing assay Asshown in Fig 4a c although the higher number ofmigration cells was observed in the TGF inducedHepG2 cells as compared to controls the addition ofHHT reduced the migrated cells Importantly concurrenttreatment with HHT and NVPBHG712 a small molecule EphB4 kinasespecific inhibitor had a greaterrestraint effect on the migration of TGF inducedHepG2 cells Wound healing assay showed similar resultsthat HHT could delay the closure of wound gaps in TGF induced HepG2 cells whereasthe addition ofEphB4 siRNA impaired such effect Fig 4b d Theseresults indicated that TGF induced the migration abilityin HepG2abrogated byEphB4 suppression of HHTcells which could beHHT bound to EphB4 and suppressed its expressionWe further evaluated the regulation of HHT on EphB4expression The results showed decreased EphB4 proteinexpression after HHT treatment both in HepG2 cells andtumor tissues Figs 5a c and S2a b Exogenous stimulation with soluble EphrinB2 Fc increased EphB4 proteinexpression while in HepG2 cells treated with EphrinB2 Fcand HHT the protein levels of EphB4 were strikinglydecreased Figs 5b and S2c HHT treatment resulted in aremarkably reduced EphB4 mRNA level at a dosedependent manner Figs 5d and S2d We treatedHepG2 cells with NVPBHG712 HHT or both to evaluate the change of EphB4 expression The results indicated that coadministration of HHT and NVPBHG712produced an even greater decrease in the expression levelof EphB4 in HepG2 cells than by either alone Figs 5e andS2e Given these findings a molecular docking assay wasconducted to confirm the affinity of HHT bound to theactive site of EphB4 The results revealed that HHToccupied in the active site of EphB4 through five hydrogen bonds which were associated with amino acid residues LYS647 GLU664 TYR736 ASP758 and THR Fig 5f The molecular docking results indicated thatHHT fit well with EphB4EphB4 was positively correlated with catenin in HCCpatients and HHT inhibited the phosphorylation andnuclear translocation of cateninEpithelial to mesenchymal transition is a prerequisitefor cell migration and lies downstream of catenin23Although previousstudies have reported that EphOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT suppressed HepG2 cell migration induced by TGF stimulation a Transwell assays were conducted to observe the migratory cellsin control and TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 treated HepG2 cells Scale bars μm b The migrationrate of control and TGF TGF HHT or TGF HHT EphB4 siRNA treated HepG2 cells observed through woundhealing assays Scale bars μm c Quantification of a n d Quantification of b n All data represent mean ± SEM p p and p compared tothe indicated groupsreceptor is conducive to EMT progression in hepatomacells24 the relationship between EphB4 and catenin hasnever been shown before To investigate the role ofcatenin in HCC we analyzed the mRNA level ofcatenin in HCC patients using The Cancer GenomeAtlas TCGA database RNASeq data from this databaseshowed that catenin expression was significantly higherin carcinoma tissue compared with paracarcinoma tissueFig 6aImmunohistochemistry was used to detectcatenin expression in pairs of HCC and noncarcinoma tissues The results showed that cateninexpression was remarkably increased in carcinoma tissuescompared with noncarcinoma tissues Fig 6b c whichwas consistent with the findings in TCGA database Todelineate the possible relationship between EphB4 andcatenin Spearman™s correlation analysis was conductedand the results revealed that catenin expression waspositively correlated with EphB4 levels in HCC tumortissues Fig 6dOfficial journal of the Cell Death Differentiation AssociationWe next analyzed the regulation of catenin in HepG2cells exposed to HHT Western blot analysis indicatedthat HHT could downregulate catenin expression andupregulate the phosphorylation of catenin level both inHepG2 cells and xenograft tumors Figs 6e f and S2f gThese results were also observed in immunohistochemicalassay for xenograft tumors Fig 6g And a remarkablyreduced catenin mRNA level was also observed inHHTtreated HepG2Immunofluorescence staining was used to examine the distribution of catenin in HepG2 cells exposed to HHT Theresults in Fig 6h demonstrated that HHT restrained thelevel of catenin in the nucleus as well as in the cytoplasm Figure 6eshowed that phosphorylation ofcatenin was obviously increased at and nM ofHHT which has been reported to contribute to process ofcatenin degradation25 These data indicated that HHTsuppressed nuclear translocation of catenin and promoted its phosphorylationS2hcellsFig 0cZhu Cell Death and Disease Page of Fig HHT bound to EphB4 and suppressed its expression a Western blot analysis of HepG2 cells EphB4 expression after HHT treatmentb Western blot analysis of EphB4 expression in HepG2 cells treated with HHT orand EphrinB2 Fc c Immunochemistry analysis of EphB4 expression inHepG2 tumors after HHT treatment n — magnification d RTPCR analysis of HepG2 cells EphB4 expression after HHT treatment n Alldata represent mean ± SEM p compared to vehicle controls e Western blot analysis of HepG2 cells EphB4 expression after HHT NVPBHG712or both treatments f Molecular docking analysis of the EphB4 protein and HHTEcadherin was overexpressed in HCC patients and HHTregulated EMTrelated moleculesGiven the positive correlation of EphB4 and cateninin HCC patients the Ecadherin expression in HCCpatients was examined As shown in Fig 7a b lowerEcadherin protein was observed in carcinoma tissueswith higher expression in the noncarcinoma tissue groupBased on the result that Ecadherin was reduced in HCCpatients and HHT could restrain the migration of HCCcells we next analyzed the effect of HHT on EMTrelatedmolecules by western blotting and immunohistochemistryassay Promotion of Ecadherin and inhibition of Snailwere observed in HHTtreated HepG2 cells and tumorsFigs 7c d g and S3a b Furthermore the results in Figs7e“g and S3c d indicated that the essential members ofMMPs family MMP2 MMP3 and MMP9 were suppressed by HHT both in HepG2 cells and in the tumortissues of xenograft models And the mRNA level ofMMP2 and MMP9 were reduced in a dose depensentmanner in HepG2 cells exposed to HHT Fig S3eHHT repressed catenin and EMTrelated moleculesthrough EphB4 suppressionNext the expression changes of EphB4 catenin andEMTrelated molecules after HHT administration for thedifferent time points were evaluated by western blottingThe results in Figs 8a and S4a“c demonstrated that theprotein level of EphB4 was significantly decreased afterHHT treatment within h and the expression of othermolecules was unchanged attime point ThethisOfficial journal of the Cell Death Differentiation Associationexpression and phosphorylation of catenin wereremarkably changed within h of HHT administrationIncreased Ecadherin expression and decreased SnailMMP2 and MMP9 expression were observed within hof HHT treatment These findings indicated that HHTmight regulate the expression of catenin and EMTrelated molecules by targeting EphB4 receptor NVPBHG712 was utilized to investigate the changes in thesemolecules after EphB4 suppression The results in Figs 8band S4e demonstrated that both HHT and NVPBHG712could suppress catenin expression and promote itsphosphorylation level Furthermore the upregulation ofEcadherin and downregulation of Snail MMP2 andMMP9 were also seen in HHT or NVPBHG712 monotherapy These effects exerted by a single administrationof HHT or NVPBHG712 were significantly augmentedby the combination of the two moleculesEMTrelated molecules in HepG2 cells following TGF stimulation was also investigated by western blot assayand the results were shown in Figs 8c and S5a b Theexpression of Ecadherin was downregulated and theprotein levels of Snail MMP2 and MMP9 were upregulated by TGF and these effects could be reversed by theaddition of both HHT and NVPBHG712 And concurrent addition of HHT and NVPBHG712 furtheraugmented the effect of monotherapyDiscussionContinuous stimulation of proliferative signals andmaladjustment of related monitoring mechanisms are 0cZhu Cell Death and Disease Page of Fig EphB4 was positively correlated with catenin in HCC patients and HHT inhibited the phosphorylation and nuclear translocation ofcatenin a mRNA expression of EphB4 in HCC carcinoma tissue and paracarcinoma tissue in the TCGA database p b Representativefigures of immunohistochemical analysis of catenin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCC patients respectively — magnification c Quantification of b n p d The positive correlation between the expression ofcatenin and EphB4 e Protein expression of catenin and pcatenin in HepG2 cells treated with HHT for h f Protein expression of cateninand pcatenin in HepG2 tumor EphB4 expression after HHT treatment g Immunochemistry assay of catenin and pcatenin in HepG2 tumortissues — magnification h Immunofluorescence analysis of the catenin protein in HepG2 cells treated with HHT catenin green DAPI bluestaining and merged images indicate the nuclear translocation and expression of catenin Scale bars μm All data represent mean ± SEMimportant causes of tumor formation The growth factorreceptor can be activated by growth factors to generateintracellular cascade signals to regulate the proliferationof tumor cells EphB4 is an important member of thereceptor tyrosine kinase family which is overexpressedand conduces to tumor growth and migration in variouscancers61315 Our previous study has confirmed theoverexpression of EphB4 in the tumor tissues of HCCpatients emphasizing EphB4 a potential target for HCCtreatment17 However there is no drugs targeting EphB4on the market In this study we found the inhibitory effectof HHT on HCC cell proliferation and migration in anEphB4 dependent manner and the underlying preliminarily mechanism was clarifiedHHT has been proved effective in the treatment ofleukemia butin HCC inhibition wasunknown We revealed the antiproliferative ability ofits potentialHHT on several HCC cell lines In particular HepG2cells with the highest EphB4 protein expression were themost sensitive to HHT treatment demonstrating thatthe inhibitory effect of HHT on HCC cells might berelated to EphB4 expression Xenograft models in nudemice confirmed the inhibitory effect of HHT on HepG2cell growth in vivo For in vitro experiments EphB4overexpression and EphrinB2 Fc stimulation increasedthe sensitivity of wild type HepG2 or Hep3B cells toHHT while transient transfection with EphB4 siRNAdecreased such effect in HepG2 cells Similar resultswere drawn from in vivo experimentsthat HHTexhibited enhanced inhibitory effect in xenograft ofEphB47721 cells compared to xenograft of wild type cells The results above indicated that EphB4might play an indispensable role in the suppression ofHCC cell proliferation by HHTOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Ecadherin was overexpressed in HCC patients and HHT regulates EMTrelated molecules a Representative figures ofimmunohistochemical analysis of Ecadherin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCCpatients respectively — magnification b Quantification of a n p All data represent mean ± SEM c Protein expression of Ecadherin and Snail in HepG2 cells treated with HHT for h d Protein expression of Ecadherin and Snail in HepG2 tumor EphB4 expression after HHTtreatment e Protein expression of MMP2 MMP3 and MMP9 in HepG2 cells treated with HHT for h f Protein expression of MMP2 MMP3 andMMP9 in HepG2 tumor tissues after HHT treatment g Immunochemistry assay of Ecadherin MMP2 and MMP9 in HepG2 tumor tissues —magnificationInvasion and migration are the main causes of tumormetastasis and the critical juncture of tumor staging inHCC2627 Since EphB4 has been reported with promotingcell migration potentialin both normal and malignantcells7 we investigate the role of EphB4 in cell migrationsuppression in HHTtreated HCC cells Our results indicated that both EphB4 overexpression and exogenous stimulation with soluble EphrinB2 Fc exacerbated theantimigratory ability of HHT on SMMC7721 cells both inwound healing and transwell migration assay FurthermoreTGF a multifunctional cytokine was used to stimulatethe migration ability of HepG2 cells28 The obtained resultsdemonstrated that HHT restrained the migration of HepG2cells stimulated by TGF while EphB4 knockdown bysiRNA impaired such inhibitory effect Combined HHT andNVPBHG712 treatment significantly augmented the antiin TGFstimulated HepG2 cells asmigratory effectcompared to either agent alone Our further investigationconfirmed that HHT was able to bind to EphB4 withhydrogen bonds and suppress its expression both in vitroand in vivo These results indicated that HHT could inhibitcell migration by regulating EphB4 in HCCOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT repressed catenin and EMTrelated molecules through EphB4 suppression a Protein expression of catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either HHT nM NVPBHG712 μM or the combination of both b Proteinexpression of EphB4 catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with HHT nM for the indicated durationc Protein expression of EphB4 Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either vehicle TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 d Schematic diagram of HHT inhibited the migration of HCCIt has been reported that Eph receptor could mediateEMT progression and adhesion to conduce migratory andmetastatic processes in hepatoma cells24 There is a wideacceptance that EMT is a prerequisite for cell migrationand catenin can trigger EMT2329 yet whether EphB4could regulate catenin remains unknown catenin wasthe key molecule of the Wntcatenin pathway and thenuclear translocation of which could not only promotethe expression of matrix metalloproteinases MMPs butalso suppress Ecadherin expression3031 In this studyboth TCGA database and our own HCC patient samplesanalysis demonstrated that catenin was significantlyoverexpressed in HCC patients at protein and mRNAlevels We also analyzed the expression data of EphB4 andcatenin in TCGA database and the results indicated thatthe mRNA level of the two molecules in HCC was significantly correlated suggesting that catenin might playa critical role in HCC migration suppression by HHT Weexamined the regulation of HHT on catenin and theresults showed that HHT strikingly inhibited cateninexpression at protein and mRNA level and promoted itsphosphorylation in vitro and in vivo Moreover the resultof immunofluorescence assay showed that the nucleartranslocation of catenin was restrained by HHTAs a key molecule of tumor migration Ecadherincould be regulated by catenin and the loss of Ecadherin is the critical marker of EMT2329 Wecompared the protein expression of Ecadherin in thecarcinoma tissues of HCC patients and the noncarcinoma tissues The resultindicated that Ecadherin level was prominently decreased in the carcinoma tissues compared to that in the noncarcinomatissues HHT treatment upregulated the protein levelof Ecadherin both in HepG2 cells and xenografttumors Furthermore Snail is a transcription factorand its expression is increased during the process ofOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of EMT We found that Snail expression wassignificantly downregulated in HHTtreated cells andtumors Most of the primary tumor cells are polarepithelial cells and connected to each other by intercellular adhesion molecules As the tumor progressesthe intercellular adhesion molecules are degraded byMMPs15 Tumor migrationrelated molecules MMPsare the downstream signaling of the Wntcateninpathway and could be regulated by catenin Thisstudy indicated that the expression of MMPs including MMP2 MMP3 and MMP9 was significantlysuppressed by HHT in vitro and in vivoThe obtained data showed that HHT could targetEphB4 and suppress its expression The expression ofEphB4 and catenin in HCC was positively correlatedaccording to TCGA data analysis HHT treatmentregulated the expression of catenin and its downstream signaling such as Ecadherin and MMPs Nextwe focused on the relationship between the effect ofHHT on EphB4 and catenin and the downstreamsignaling We investigated the protein levels in HepG2cells exposed to HHT for different duration and theresults confirmed that catenin might be the downstream signaling of EphB4 receptor EphB4 specificinhibitor NVPBHG712 could suppress the proteinlevel of catenin and promote its phosphorylationThe expression of Ecadherin Snail and MMPs wasalso significantly changed after EphB4 was suppressedby NVPBHG712 And the regulating effect on EphB4catenin and its downstream cascades was remarkablycoadministration of HHT and NVPBHG712 In addition the increased expression of Snail and MMPs anddecreased expression of Ecadherin confirmed thatTGF induced EMT in HepG2 cells Both HHT andNVPBHG712 could reverse the regulating effect ofTGF and such effect was enhanced by combinedHHT and NVPBHG712 treatment These findingsindicated that HHT could reverse the EMT of HepG2cells by restraining EphB4 expression the suppressionof which further inhibited the nucleus translocation ofcatenin and regulated the expression of EMT related molecules including Ecadherin Snail MMP2and MMP9in HepG2augmentedcellsafterIn conclusion we discovered a positive correlation ofEphB4 and catenin in HCC patients and that EphB4 wasinvolved in HCC cell migration progression by regulatingcateninmediated EMT HHT suppressed EphB4expression and further led to catenin loss resulting inthe regulation of Ecadherin Snail and MMPs to preventEMT progression in HCC cells Fig 8d Our researchmay provide new insight into the migration mechanism ofEphB4 in HCC and HHT possesses great potential in thedevelopment of antiHCC drugsOfficial journal of the Cell Death Differentiation AssociationMaterials and methodsReagentsHHT HPLC ‰¥ was obtained from Baoji Herbest Biotech Co Ltd Shaanxi China NVPBHG712Purity ‰¥ was purchased from MedChemExpressCo Ltd Dulbecco™s modified Eagle™s mediumDMEM RPMI medium and PBS were fromHyClone Logan UT USA Fetal bovine serum FBSwas purchased from ExCell Bio Co Ltd ShanghaiChina MTT powder RNase and propidium iodidewere from Sigma“Aldrich St Louis MO USADimethyl sulfoxide DMSO was from Tianjin KemiouChemical Reagent Co Ltd Tianjin China OptiMEM medium was purchased from Gibco CaliforniaUSA Trypsin and PenicillinStreptomycin solutionwere obtained from Beijing Solarbio Science Technology Co Ltd Beijing China Lipofectamine reagent was purchased from Invitrogen Carlsbad CA USA RIPA Lysis Buffer and BCA proteinassay reagent kit were purchased from Pioneer Biotechnology Co Ltd Protease and phosphatase inhibitors were obtained from Roche TechBaselSwitzerland Ultra Signal Enhanced Chemiluminescent ECL Reagent kit was purchased from 4A Biotech Co Ltd Beijing China EphB4 catenin andpcatenin rabbit mAb were obtained from CellSignaling Technology Boston MA USA Ecadherin Snail GAPDH rabbit mAbs and goat antirabbitIgG were purchased from Protein technology GroupChicago Illinois USA MMP2 MMP3 and MMP9rabbit mAb were from ABclonal Boston MA USAThe EphB4 bacterial strain was from VectorBuilderPatientsAll the patients who were eligible underwent surgery atthe First Affiliated Hospital of Xi™an Jiaotong UniversityFifteen HCC tissues from HCC patients and fifteenhepatic tissues from nonHCC patients were obtainedfrom consenting patients and used with permission ofbiomedical ethics committee of Xi™an Jiaotong UniversityHealth Science Center Project No Cell cultureHuman hepatocellularcarcinoma HepG2 Hep3BSMMC7721 Bel7402 and Bel7404 cells werepurchased from the Shanghai Institute of Cell Biology atthe Chinese Academy of Sciences Shanghai Chinawithout mycoplasma contamination The HepG2 andHep3B cells were cultured in DMEM with FBS and Penicillin and Streptomycin solution SMMC7721Bel7402 and Bel7404 cells were cultured in RPMI1640with FBS and Penicillin and Streptomycin solution Allthe cells were incubated in a humidifiedatmosphere incubator of CO2 at °C 0cZhu Cell Death and Disease Page of Cell viability assayMTT method was used to analyze cell viability Thegrowing cells were seeded in 96well plates overnightThen the cells were treated with increased concentrationof HHT for h followed by incubation with the mixtureof serum free medium and MTT solution for “ h Themixture was replaced by DMSO After min shakingthe plates were determined using EPOCH BioTekInstruments Inc USA at a wavelength of nmColonyforming assayThe growing cells were seeded in 12well plates at adensity of cells per well Following h incubationthe cells were exposed to HTT for h followed bycultured in fresh complete medium for weeks Afterwashed twice with PBSthe colonies were fixed bymethanol and stained using crystal violet Imageswere obtained via an inverted fluorescence microscopeMigration assayThe HCC cells were cultured into the upper chamber at adensity of — cells per well accompanied by μLcomplete medium in the lower chamber Following incubation for h for EphB4 p

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults™ activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged “ years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchison™sperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[“] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [“] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was ˆ’ °c lowest ˆ’ °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [“] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as œnonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb ‰ mets lpa“ mets and mvpa ‰¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package œcompositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemar™s test wasused to compare nonsnowfall and snowfall season™sproportions of those adhering to physical activity guidelines ie ‰¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of “ min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchison™s perturbation method [“] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained‰¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participant™s characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold “ yrsoldold ‰¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ‰¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [“] data where levels of adults™ physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [“ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors™ contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from ˜the policy research institute ministryof agriculture forestry and fisheries in japan™ and ˜the pfizer healthresearch foundation™ and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian government™s operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo “ japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet “kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk ofbreast cancer colon cancer diabetes ischemic heart disease and ischemic 0camagasa bmc public health page of stroke events systematic review and doseresponse metaanalysis for theglobal burden of disease study bmj 2016354i3857aune d norat t leitzmann m tonstad s vatten lj physical activity andthe risk of type diabetes a systematic review and doseresponse metaanalysis eur j epidemiol “arem h moore sc patel a hartge p berrington de gonzalez avisvanathan k campbell pt freedman m weiderpass e adami ho leisure time physical activity and mortality a detailed pooled analysis of thedoseresponse relationship jama intern med “sofi f valecchi d bacci d abbate r gensini gf casini a macchi c physicalactivity and risk of cognitive decline a metaanalysis of prospective studiesj intern med “young dr hivert mf alhassan s camhi sm ferguson jf katzmarzyk ptlewis ce owen n perry ck siddique j yong cm sedentary behavior andcardiovascular morbidity and mortality a science advisory from theamerican heart association circulation biswas a oh pi faulkner ge bajaj rr silver ma mitchell ms alter dasedentary time and its association with risk for disease incidence mortalityand hospitalization in adults a systematic review and metaanalysis annintern med “ding d lawson kd kolbealexander tl finkelstein ea katzmarzyk pt vanmechelen w pratt m the economic burden of physical inactivity a globalanalysis of major noncommunicable diseases lancet “tucker p gilliland j the effect of season and weather on physical activity asystematic review public health “ barnett dw barnett a nathan a van cauwenberg j cerin e builtenvironmental correlates of older adults' total physical activity and walking asystematic review and metaanalysis int j behav nutr phys act shephard rj tudorlocke c the objective monitoring of physical activitycontributions of accelerometry to epidemiology exercise science andrehabilitation new york springer 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validity of estimating physical activity intensity using a triaxialaccelerometer in healthy adults and older adults bmj open sport exercmed 20195e000592kurita s yano s ishii k shibata a sasai h nakata y fukushima n inoue stanaka s sugiyama t comparability of activity monitors used in asianand westerncountry studies for assessing freeliving sedentary behaviourplos one 201712e0186523 murakami h kawakami r nakae s nakata y ishikawatakata k tanaka smiyachi m accuracy of wearable devices for estimating total energyexpenditure comparison with metabolic chamber and doubly labeledwater method jama intern med “tudorlocke c camhi sm troiano rp a catalog of rules variables anddefinitions applied to accelerometer data in the national health andnutrition examination survey prev chronic dis 20129e113troiano rp berrigan d dodd kw masse lc tilert t mcdowell m physicalactivity in the united states measured by accelerometer med sci sportsexerc “ haskell wl lee im pate rr powell ke blair sn franklin ba 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inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4“ while targeted analyses in larger cohorts have confirmed the prevalence of specific driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have confirmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9“ However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of — distinct coverageof — generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in “ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identified in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of —distinct coverage of — Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to confidently identify even subclonal alterations in sampleswith low neoplastic purity see œMethods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentified an average of somatic mutations in samples fromnoninvasive components range “ and an average of somatic mutations in invasive carcinoma samples range“Fig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentified an average of five shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1“S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identified in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2ˆ’ˆ’ˆ’SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identified in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61Hˆ’1225380275_TGKRASQ61Hˆ’1225380275_TAKRASG12VTMEM56ˆ’RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ˆ’ABRF645SALPK1R873RLOHchrX275ˆ’DELchr9218ˆ’2377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716ˆ’LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725ˆ’SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123ˆ’LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3cˆ’2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546ˆ’DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identified in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identified in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we confirmed the high prevalenceof mutations in previously identified pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identified in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identified somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identified alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentified somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Amplifications of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with biallelicloss”theATM lossand cancerspecific biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspecific genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations confined to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identified driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking finding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentified two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identified in the matched noninvasiveand invasive samples the copy number profiles were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identified samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identifiedmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identified IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identified an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedfive different samples from invasive cancer”three samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the specific invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identified in almost every case SupplementaryFig S19 However we also identified signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deficiency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeœMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI “ years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI “ years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679“ However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identified somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identified alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identified in ATM are likely to be bona fidesomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identified in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21“The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24“We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspecific mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspecificSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identified a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have specificallydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number profilesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identified multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identified multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32“ RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci

Despite the proven benefits of iron chelation therapy ICT in the management of chronic iron overload and related complications compliance to longterm ICT is challenging Results from the ECLIPSE study an label randomized multicenter 2arm phase study evaluated the safety of deferasirox dispersible tablet and filmcoated tablet FCT formulations in patients with transfusiondependent thalassemia TDT or very low low or intermediate risk myelodysplastic syndrome MDS treated over weeksMethods The aim of the current study a 2year label multicenter singlearm phase study is to evaluate the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or lowerintermediaterisk MDS treated for years after the completion of weeks of treatment with deferasirox in the ECLIPSE phase studyResults Of patients enrolled completed treatment and study Adverse events AEs reported in most patients were of mild to moderate severity Headache and diarrhea were the most frequently reported AEs None of the serious AEs including death were considered treatment related No new safety signal was identified and longterm safety of deferasirox FCT was consistent with the known safety profile of deferasirox No major concerns associated with gastrointestinal tolerability renal safety or hematological abnormalities thrombocyt ianeutr ia were reported during the years Patients receiving deferasirox FCT had a treatment compliance by pill count of and persistence continuous use for ‰¥ days of Reduction in serum ferritin level was almost consistent starting from week across all postbaseline time points relative reduction month month Correspondence giovanbattistaruffoarnascivicoit UOC Ematologia e Talassemia AO CivicoDi CristinaBenfratelli Piazza Nicola Leotta Palermo ItalyFull list of author information is available at the end of the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTartaglione a0et a0al Exp Hematol Oncol Page of Conclusions The results from this 2year interventional study suggest that the recommended dosing of deferasirox FCT with better tolerability palatability and compliance offers a favorable option of ICT for longterm management of iron overload and associated complications in TDTTrial registration ClinicalTrialsgov NCT02720536 Registered March wwwclini caltr ialsgovct2showNCT02 Keywords Deferasirox Chelation Filmcoated tablet Iron overload Longterm Safety Transfusiondependent thalassemia Myelodysplastic syndromeIntroductionIron overload in chronic anemias represents a serious consequence of impaired hematopoiesis and repeated blood transfusions leading to endan damage reduced quality of life and decreased survival Iron chelation therapy ICT can be a lifelong requirement in chronic transfusiondependent refractory anemias including thalassemia sicklecell disease and myelodysplastic syndrome MDS [ ] Despite the proven benefit of ICT patient compliance to longterm ICT is challenging [] Compliance with ICT is reported to influence the frequency and severity of iron overload“related complications [“] and a substantial increase in morbidity mortality and treatment cost has been seen among patients who are noncompliant to ICT []Currently main iron chelators are available for clinical use deferoxamine deferiprone and deferasirox Parenterally administered deferoxamine was the mainstay of chelation therapy until the availability of oral chelators in [] Oncedaily oral administration of deferasirox dispersible tablet DT formulation presented a better option with greater compliance and quality of life over parenteral deferoxamine [] Currently there are no direct comparison studies for the oral chelators however oncedaily simpler use of deferasirox has been projected to be a more costeffective option than the thricedaily administration of oral deferiprone in the management of longterm complications [ ] The deferasirox filmcoated tablet FCT formulation with a simpler oral administration and improved palatability and gastrointestinal GI tolerability compared with deferasirox DT offers a better option for optimal patient acceptance and improved compliance to longterm therapy [ ] Thus an appropriate choice of ICT plays an important role in patient compliance to chelation therapyThe deferasirox FCT used in this study contained the same active substance of the iron chelator deferasirox and was strengthadjusted to achieve comparable exposure to the currently approved deferasirox DT Deferasirox FCT is available in dose strengths a0mg a0mg and a0mg and is dosed based on body weight Unlike the DT deferasirox FCT can be administered once daily orally without any need for dispersion either on an empty stomach or with a light meal [ ]Results from the ECLIPSE study an label randomized multicenter 2arm phase study that evaluated the safety of deferasirox DT and FCT formulations in patients with transfusiondependent thalassemia TDT or MDS very low low or intermediate risk treated over a0weeks have been published previously [] The purpose of the current study was to collect data on the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or very low low or intermediaterisk MDS who had the possibility to continue treatment with deferasirox FCT after completion of a0 weeks of treatment in the ECLIPSE study The study also aimed to collect efficacy data for deferasirox FCT in the reduction or maintenance of iron burden as measured by the serum ferritin levelMethodsKey inclusion and a0exclusion criteriaPatients recruited at European sites [Austria n Greece n Italy n ] who had completed the 24week treatment in the ECLIPSE study with tolerance to deferasirox and fulfilled all eligibility criteria were included in this study Patients were male or female aged ‰¥ a0 years with TDT or very low low or intermediaterisk MDS who had received deferasirox DT at doses ‰¥ a0mgkgday or ‰¥ a0mgkgday respectively as per clinical judgement Patients had a transfusion history of ‰¥ packed red blood cell units were anticipated to be transfused with ‰¥ a0unitsyear during the study and had serum ferritin a0ngmL at screening Key exclusion criteria in the study included patients with creatinine clearance CrCl below contraindication limit as per local label serum creatinine SCr — upper limit of normal ULN alanine aminotransferase ALT — ULN unless liver iron concentration was confirmed as a0mg Fedry weight within a0months prior to screening urine protein to creatinine ratio UPCR a0mgmg impaired GI function that may significantly alter the absorption of oral deferasirox or clinicallaboratory evidence of active hepatitis Bhepatitis C infection 0cTartaglione a0et a0al Exp Hematol Oncol Page of Study designThis was a 2year label multicenter singlearm phase study NCT02720536 aimed to provide additional efficacy safety and drug exposure data following the ECLIPSE study []Patients who were assigned to either the deferasirox DT or the deferasirox FCT arm and had completed the study period of a0weeks with tolerance to deferasirox in the ECLIPSE study were allowed to participate in this study It was planned that any patient continuing directly from the ECLIPSE study would receive the same dose of deferasirox FCT or an equivalent FCT dose at the start of this study corresponding to their DT dose at the end of the ECLIPSE study As all patients had a lag period between the completion of the ECLIPSE study and the start of this study ie patients who completed the ECLIPSE study switched to commercially available deferasirox DT or another ICT they entered the current study following a washout period with a starting dose that was based on clinical judgment For each patient the daily dose was calculated based on the patient™s actual body weight and then rounded up or down to the nearest whole tablet according to available strengths of deferasirox FCT tablets a0mg a0mg and a0mg Dose adjustments were allowed every a0months based on serum ferritin levels and clinical judgment with ± to a0mgkgday up to a maximum dose of a0mgkgdayThe study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki and was approved by independent ethics committees at participating sites The study is registered at wwwclini caltr ialsgovct2showNCT02 Patients or parentsguardians provided written informed consent or assent prior to enrollmentOutcomesThe primary objective of the study was to evaluate the overall safety of the deferasirox FCT formulation measured by the frequency and severity of adverse events AEs and changes in laboratory values of interest such as SCr and CrCl The secondary objective was to evaluate the efficacy of deferasirox FCT with respect to serum ferritin levels decreased or maintained according to individual therapeutic goal measured by the absolute and relative changes in serum ferritin levels over timeStatistical evaluationsNo formal sample size was calculated A maximum of patients aged ‰¥ a0years were originally planned to be enrolled from the ECLIPSE study patients were enrolled into this study There were no screening failuresData from all centers participating in this study were for analyses The statistical software SAS® version collected using electronic case record forms and pooled was used for analysis The analyses were descriptive in nature no hypothesis was tested Data were summarized with respect to demographic and baseline characteristics primary and secondary assessments along with safety observations All analyses were based on data collected as per protocolscheduled assessments according to or including clinical judgment of the investigatorPatient compliance to deferasirox FCT was evaluated using the count of deferasirox FCT by the relative consumed tablet count Descriptive statistics including confidence intervals CIs for the mean relative consumed tablet counts were provided Persistence defined as continuous use of deferasirox FCT without a gap for ‰¥ or ‰¥ a0days over a fixed time interval of interest was summarized at month a0 month month and month The incidence of any treatmentemergent AEs ie AEs from the start of study treatment to a0days after the last intake of study drug overall and by maximum grade severity mild moderate or severe as reported by the clinician were summarized using frequency counts and percentages of patients For each of the laboratory parameters of interest SCr CrCl ALT and aspartate aminotransferase [AST] the worst postbaseline values were summarized as shift tables based on notableextended ranges For serum ferritin and hematological parameters red blood cells [RBCs] platelets total white blood cells hemoglobin and hematocrit the absolute mean and the relative mean changes from baseline were summarized at each postbaseline visitResultsOverall patients were enrolled from countries across Europe of whom discontinued early from treatment Fig a0Of the patients enrolled the majority patients [] including patients aged a0 years had TDT and patients had MDS all aged ‰¥ a0years Most patients were Caucasians and females comprised of the study population All patients had a history of prior ICT Demographics and other baseline characteristics are described in Table a0In most patients deferasirox monotherapy was the last ICT before study treatment The majority of patients had received prior medication or therapy The most common prior medications by the Anatomical Therapeutic Chemical classification system class included vitamin D and vitamin D analogues patients [] proton pump inhibitors PPIs patients [] sequential preparations of progestogens 0cTartaglione a0et a0al Exp Hematol Oncol Page of Patients enrolled N53Countries Austria Greece Italy Disease TDT MDS Completed treatment and studyaDiscontinued from treatment Withdrewconsent Pregnancy Unsatisfactorytherapeuticeffect Death Adverseevents Abnormallaboratoryvalues Unwillingnessto comply withprocedures Fig Patient disposition MDS myelodysplastic syndrome TDT transfusiondependent thalassemia aThe patients who completed the study had received treatment for at least monthsand estrogens patients [] fixed combinations of progestogens and estrogens patients [] and thyroid hormones patients []Almost all patients [] received concomitant medication or significant nondrug therapies on or after the start of study treatment The most common concomitant medications by ATC class included vitamin D and vitamin D analogues patients [] anilides patients [] antibiotics patients [] other agents for local oral treatment patients [] PPIs patients [ and other antibiotics for topical use patients []All patients received at least transfusions during the study with a maximum of transfusions received by patient The majority of patients [] including the MDS patients received to transfusions Additional file a0 Table a0S1Exposure to a0treatment and a0complianceThe mean standard deviation [SD] duration of exposure to treatment was days with most patients [] being treated for at least a0weeks The majority of patients receiving deferasirox FCT were in the longest exposure category ‰¥ a0weeks The sum of each patient™s treatment exposure to deferasirox FCT was patientyears Almost all patients [] received deferasirox FCT at an average dose of ‰¥ a0 mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose received Table a0 MDS patients n received an average dose of ‰¥ a0mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose receivedPatients had a mean relative consumed tablet count of CI “ The proportions of patients with continuous use of deferasirox FCT with no interruption for ‰¥ a0days and ‰¥ a0days were n and n respectively at a0monthsEfficacyA decrease in mean serum ferritin levels was observed from week a0 onward except for month a0 though the median serum ferritin levels showed a consistent decrease across all postbaseline time points The mean SD actual decrease in serum ferritin level was µgL from baseline to month a0 µgL from baseline to month a0 and µgL from baseline to month a0 Fig a0 The decrease relative change in [SD] was higher at month a0 than at month a0 and month a0 [] vs [] vs [] The mean SD actual decrease in serum ferritin level from baseline to month a0 was reported as a0µgL relative change in percentage in patient at month a0 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Demographics and a0other baseline characteristicsDemographic variableAge years mean SDAge category years n ‰¥ to ‰¥ to ‰¥ 65aSex n Male FemaleBMI kgm2 mean SDPredominant race n Caucasian Asian OtherbMain underlying disease n MDS IPSSR risk stratification Very low risk Low risk Intermediate risk TDTTime since diagnosis years mean SD MDS TDTPrior ICT received during the ECLIPSE study n Deferasirox DT Deferasirox FCTLast ICT received before deferasirox FCT in the current study n Deferiprone Deferoxamine and deferiprone Deferasirox monotherapyTransfusion history Total number of transfusions received over the past months mean SD Time since last blood transfusion days mean SDBaseline serum ferritin µgL mean SDDeferasirox FCT N BMI body mass index DT dispersible tablet FCT filmcoated tablet ICT iron chelation therapy IPSSR Revised International Prognostic Scoring System MDS myelodysplastic syndrome SD standard deviation TDT transfusiondependent thalassemiaa The oldest patient was years oldb Other included two patients who selfidentified as whiteSafetyAdverse eventsOf the patients almost all [] reported at least AE regardless of study drug relationship Additional file a0 Table a0S2 provides an overview of all AEsThe most common AEs by preferred term were headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain The most common AEs reported in of patients by system an class SOC during the treatment with deferasirox FCT were related to infections and infestations [] influenza rhinitis gastroenteritis pharyngitis and urinary tract infection occurred in of patients by preferred term followed by GI disorders [] diarrhea nausea vomiting upper abdominal pain and abdominal pain occurred in of patients by preferred term Table a0 None of the AEs in the infections and infestations SOC were treatment related Of the AEs that were suspected to be treatment related in patients increased UPCR 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Exposure to a0treatment and a0complianceDeferasirox FCT N Duration of exposure days mean SDDuration of exposure categories weeks n to to to ‰¥ Patient treatment yearsAverage actual dose mgkgday mean SDAverage actual dose category mgkgday n to to ‰¥ Compliance Relative consumed tablet count mean SD Persistence Continuous use of deferasirox FCT with no interruption for ‰¥ days n Up to months n Up to months n Up to months n Up to months n Continuous use of deferasirox FCT with no interruption for ‰¥ days n Up to months n Up to months n Up to months n Up to months n FCT filmcoated tablet SD standard deviation diarrhea increased blood creatinine gastritis and proteinuria were the most common reported in of patients AEs by preferred term Of these suspected treatmentrelated AEs increased blood creatinine and diarrhea both of mild severity were reported in MDS patientsThe maximum grade of severity of AEs was reported as mild moderate and severe in and of patients respectively Table a0 Severegrade AEs reported by preferred term irrespective of study drug relationship included increased UPCR splenomegaly atrial fibrillation cardiac failure goiter cholestasis hepatic failure gastroenteritis lower respiratory tract infection lymph gland infection urosepsis femur fracture spinal fracture lumbar vertebral fracture rib fracture ulna fracture transfusion reaction arthralgia back pain malignant melanoma papillary thyroid cancer headache device failure renal colic calculus urinary hydronephrosis and ureterolithiasis each None of the patients had severe GI AEs Moderate and severegrade AEs by maximum severity grade were reported in patients and patients with deferasirox as prior chelation therapy and patients and patients with other ICT as prior chelation therapy respectively Of the patients moderategrade AEs were reported in patients in the to a0mgkgday dailydose group and patients in the ‰¥ a0 mgkgday dailydose group while severegrade AEs were reported in patients each in the to a0 mgkgday and ‰¥ a0 mgkgday dailydose groups respectivelySerious AEs SAEs regardless of study drug relationship were reported in patients MDS n TDT n and none of these were considered treatment related SAEs reported in patients with MDS included cardiac failure device failure femur fracture cholestasis hepatic failure and malignant melanoma reported in patient who died lumbar vertebral fracture in patient and urosepsis in patient Other SAEs reported in patients with TDT included spontaneous abortion atrial fibrillation biliary colic urinary calculus cholecystitis diverticulitis fracture goiter hydronephrosis lower respiratory tract infection lymph gland infection ovarian adenoma panic attack papillary thyroid cancer renal colic rib fracture ulna fracture and ureterolithiasis None of these SAEs were reported in more than patient each Death not suspected to be treatment related occurred in a 72yearold male patient with very low“risk MDS as per the Revised International Prognostic Scoring System [IPSSR] The cause of this ontreatment death according to clinical judgment was malignant melanoma with multiple metastasis in the liver and spleen with unknown primary origin Another contributing factor for the death was liver failure and intrahepatic cholestasisAEs leading to discontinuation of study treatment were reported in patients of which drug ineffective AE in patient and serum ferritin abnormal AE in patient were considered treatment related Treatment was discontinued due to an AE moderate in severity not suspected to be treatment related in MDS patient AEs that led to dose adjustmentinterruption occurred in patients with the most frequently reported being increased UPCR patients [] and vomiting patients [] Treatmentrelated AEs that led to dose adjustmenttemporary interruption were reported in patients and included increased UPCR patients [] increased blood creatinine patients [] gastritis glycosuria proteinuria upper abdominal pain patients [] each and diarrhea gastric ulcer increased serum 0cTartaglione a0et a0al Exp Hematol Oncol Page of morfnitirrefmuresni egnahcLgµenilesabDSnaeMˆ’ˆ’ˆ’ˆ’ˆ’W2n44 M1n51M2n48M3n50 M4n43M5n46 M6n44 M7n40M8n37M9n42M10n36M11n41M12n36M13n43M14n39 M15n28M16n39 M17n32M18n31M19n31M20n33M21n26M23n28 M24n25M22n33M25n21M27n1M26n22Fig Change in serum ferritin from baseline µgL by time point M month SD standard deviation W week Error bars represent the ± SD values for the respective mean valuesNumber of observations at each timepointTime pointTable Common adverse events reported by a0maximum grade of a0severityAEs N a0System an class a0 a0Preferred termOverall n Mild n Moderate n Severe n Number of patients with at least one event Infections and infestations Rhinitis Gastroenteritis Pharyngitis Urinary tract infection Gastrointestinal disorders Diarrhea Nausea Vomiting Upper abdominal pain Abdominal pain General disorders and administration site conditions Asthenia Influenza Pyrexia Respiratory thoracic and mediastinal disorders Cough Oropharyngeal pain Investigationsa Urine proteincreatinine ratio increased Musculoskeletal and connective tissue disorders Musculoskeletal pain Nervous system disorders Headache Proportion of patients with AEs reported by preferred term and grouped by system an classAE adverse eventa Abnormal laboratory values reported as AEs 0cTartaglione a0et a0al Exp Hematol Oncol Page of ferritin hypertransaminasemia and increased transaminases patient [] each AEs required additional treatment in patients with TDT macular edema and skin ulcer in patient [] diarrhea radius fracture and breast discomfort in patient [] each and none were suspected to be treatment relatedAdverse events of a0special interestOverall patients reported AEs of special interest of which AEs suspected to be treatment related were reported in patients Common AEs of special interest incidence included increased UPCR patients [] severe and suspected to be treatment related patients [] proteinuria patients [] increased blood creatinine patients [] MDS n and hypertransaminasemia patients [] Hepatic failure due to metastatic liver disease MDS n and transfusion reaction TDT n of severe grade but not suspected to be treatment related were reported in patient each One patient with MDS discontinued the treatment due to decreased creatinine renal clearance moderate in severity but not suspected to be treatment related Additional file a0 Table a0S3Laboratory parametersWorst postbaseline elevations in SCr of ULN at consecutive measurements at least a0days apart occurred in patients MDS n TDT n One patient with MDS had worst postbaseline UPCR a0 mgmmol at consecutive measurements at least a0 days apart notable range Two patients with MDS had worst postbaseline CrCl value within the notable range a0 mLmin at consecutive measurements at least a0days apart and patients had worst postbaseline CrCl a0mLmin value Two patients with TDT had a worst postbaseline ALT level in the notable range — ULN and — baseline value Elevations of transaminases AST or ALT — ULN were Table Shift tables of a0laboratory values based on a0notableextended rangesALT UL‰¤ ULN ULN to ‰¤ — ULNTotalAST UL‰¤ ULN ULN to ‰¤ — ULNTotalSerum creatinine‰¤ ULNTotalCreatinine clearance‰¥ ‰¥ to MissingTotalUrinary proteinurinary creatinine ratio mgmmol‰¤ MissingTotalBaselinen n n n n ‰¤ ULN n ‰¤ ULN n ‰¥ n ‰¤ n Worst postbaseline value‰¤ ULN n ULN to a0‰¤ —ULN n ULN to a0‰¤ —ULN n — ULN n — ULN n Notable range n Extended range n Notable range n Extended range n Two consecutive ULN n Notable range n One value ‰¥ to a0 n Notable range n Missing n One value n Extended range n One value n Two values n Notable range n Missing n ALT alanine aminotransferase AST aspartate aminotransferase ULN upper limit of normal 0cTartaglione a0et a0al Exp Hematol Oncol Page of uncommon only patient with TDT had a postbaseline increase in AST and ALT values — ULN and — baseline value Table a0Hematological parametersThe majority of patients had low RBC of hematocrit of and hemoglobin of values at baseline Among patients with a baseline platelet count ‰¥ — 109L only patients had worst postbaseline values in the notable range ‰¥ to — 109L Similarly among patients with a baseline absolute neutrophil count ‰¥ — 109L the worst postbaseline values were found to be in the notable range ‰¥ to — 109L in patient and extended range — 109L in patient Mean ± SD change mean relative percentage change from baseline to month and month in key hematological parameters is represented in Additional file a0 Table a0S4DiscussionThis 2year phase interventional study evaluated the efficacy and safety of deferasirox FCT in adult and pediatric patients mean age a0 years with MDS n and TDT n who had previously completed a0weeks of deferasirox treatment in the ECLIPSE study over a mean duration of further a0days Almost of patients received an average actual deferasirox FCT dose of ‰¥ a0mgkgday during the study The average actual dose in MDS patients was comparatively less than that in TDT patients reflecting the notion of physicians using lower doses to treat MDS patients [] None of the patients were administered with higher than the maximum recommended doses of deferasirox FCT a0mgkgday []Various studies have demonstrated that compliance with ICT significantly improves morbidity and mortality [“ ] Compliance mean relative consumed tablet count and persistence “ rates with deferasirox FCT during this study were found to be on the higher side Persistence rates proportion of patients with continuous use of deferasirox FCT with no interruption for ‰¥ a0days or ‰¥ a0days in this study are comparable to the realworld data on persistence in patients who switched from deferasirox DT “ to deferasirox FCT “ []Common AEs reported with deferasirox FCT in this study headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain were consistent with the known profile of deferasirox [ ] AEs reported in of the patients were of mild to moderate in severity none of the GI AEs were of severe grade nor led to discontinuation of treatment These results were in line with those of previous studies which reported that GI tolerability profile may be improved with the FCT compared with the DT Lack of excipients that cause GI irritation and ease of administration along with a light meal could have contributed to the better GI tolerability of FCT [ ] It is noted that the concomitant use of PPIs during the study was higher than reported prior use This study was not designed to investigate the reasons behind the increased use of PPIs but we assume that they might have been prescribed as a general prophylaxis to avoid GI complications from other concomitant therapies analgesics antibiotics etc prescribed in comorbid conditions infections or proceduresSAEs regardless of study drug relationship were reported in of patients and none including the death due to malignant melanoma with multiple metastasis in the liver and spleen were considered treatment related Notably of SAEs reported in patients were reported in MDS patients aged ‰¥ a0 years reflecting the considerable burden of the underlying hematological disorder together with the comorbidities of the affected elderly patients [“] Discontinuation of treatment due to treatmentrelated AEs drug ineffective and abnormal serum ferritin during the a0years was observed in only TDT patients The low rate of SAEs and absence of treatmentrelated deaths in this study could have been a result of the greater compliance and persistence “ rates with deferasirox FCTThe ECLIPSE study reported that abnormal renal parametersrenal AEs were more common in patients receiving deferasirox FCT than in those receiving deferasirox DT due to an intake of higher than the recommended dose [] Most of the renal abnormalitiesAEs reported during this 2year study were mild and reversible with dosage adjustment or temporary interruption of deferasirox FCT Only patient discontinued the treatment due to decreased creatinine renal clearance which was moderate in severity and not suspected to be treatment related Increases in SCr and liver function tests in the notableextended ranges were observed in some patients and those increases were consistent with the known safety profile of deferasirox FCT [] No substantial difference in severity of all AEs was noted based on dosing groups to a0mgkgday and ‰¥ a0mgkgday as well Administration of the recommended doses of deferasirox FCT with constant dosage adjustment as per serum ferritin levels might have resulted in fewer renal and liver abnormaliti

"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowˆ’neutrophils after capecitabine therapy had adverse clinical features What™s important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowˆ’neutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowˆ’population might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [“] What™s more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[“] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [“] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhat™s more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about“ years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [“] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [“] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [“] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy [“]Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout “ years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowˆ’myeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about “ months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about “ years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15ˆ’ neutrophils CD11bCD15CD14ˆ’ or CD11bCD66bCD14ˆ’and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhat™s important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients “ months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and “ months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected “ months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16lowˆ’ group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was × Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by χ2 test The data revealed that patients inCD16lowˆ’ group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16lowˆ’ group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16lowˆ’ group while only patients wereCD16 And patients with high CA199level were found in CD16lowˆ’ group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ‰¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16lowˆ’ after therapy n nAll patients n nCD16 after therapy n nAge years‰¥GenderMaleFemaleTumor locationRectumColonTumor Size‰¥ cm cmCEA level after therapy‰¤ ngml ngmlCA199 level after therapy‰¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ‰¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16lowˆ’ group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table What™simportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about “ years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased “ months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E What™s important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ‰¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ‰¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients™ survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16lowˆ’ group p values were calculated by logrank test n in CD16 group and n in CD16lowˆ’ grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowˆ’myeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowˆ’myeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16ˆ’myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16ˆ’myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16ˆ’myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CI“““““““““p valueMultivariate analysisHR“95CI““““““““““““““p value““““““ 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16ˆ’myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16ˆ’myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16ˆ’myeloid cells we sorted peripheral blood CD11bCD16ˆ’myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16ˆ’myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in “ months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16ˆ’ in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16ˆ’myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16ˆ’myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16ˆ’myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16ˆ’N CRC R CD11bCD16ˆ’myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16ˆ’myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients “ months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearson™s correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowˆ’neutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected “ months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearson™s correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowˆ’neutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients won™t have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy What™s more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in “ months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients™ healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroup™s data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowˆ’myeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e

prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early‘onset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patients”with a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the œsilent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2“ Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance ˜RCspecific™ genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15“ A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10“ To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n ‰ a0years at diagnosis specimens collected between July “December All genetic test results from germline testing of individuals diagnosed with RC at ‰ during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of ‰ a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions “ and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PV”see below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN cˆ’ to cˆ’ MLH1 cˆ’ to cˆ’ and MSH2 cˆ’ to cˆ’ Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as ˜not reported™ Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term œrenal cancer was queried and the results were noted as œassociated with ClinVar search term ˜Renal Cancer™Statistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histology™s were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ‰¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fisher™s exact tests were used and a0pvalues ‰ were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between “ a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between “ a0years of age as compared to patients ‰ diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed ‰ Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in “ of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women ‰ in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434“After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI “ ExAc OR CI “ We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC ‰ a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC ‰ a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at ‰ a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported ‰ a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset œother based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see œMethods The groups were compared to median age at first RC diagnosis of or ‰¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1

Neurogenesis From Neural CrestCells Molecular Mechanisms in theFormation of Cranial Nerves andGangliaKarla MndezMaldonado12  Guillermo A VegaLpez34  Manuel J Aybar34 andIv¡n Velasco15 Instituto de Fisiolog­a Celular “ Neurociencias Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico Departamento de Fisiolog­a y Farmacolog­a Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autnomade Mxico Ciudad de Mxico Mexico Instituto Superior de Investigaciones Biolgicas INSIBIO CONICETUNT SanMiguel de Tucum¡n Argentina Instituto de Biolog­a œDr Francisco D Barbieri Facultad de Bioqu­mica Qu­mica yFarmacia Universidad Nacional de Tucum¡n San Miguel de Tucum¡n Argentina Laboratorio de Reprogramacin CelularInstituto Nacional de Neurolog­a y Neurocirug­a œManuel Velasco Su¡rez Ciudad de Mxico MexicoThe neural crest NC is a transient multipotent cell population that originates in thedorsal neural tube Cells of the NC are highly migratory as they travel considerabledistances through the body to reach their final sites Derivatives ofthe NC areneurons and glia of the peripheral nervous system PNS and the enteric nervoussystem as well as nonneural cells Different signaling pathways triggered by BoneMorphogenetic Proteins BMPs Fibroblast Growth Factors FGFs Wnt proteins Notchligands retinoic acid RA and Receptor Tyrosine Kinases RTKs participate in theprocesses of induction specification cell migration and neural differentiation of the NCA specific set of signaling pathways and transcription factors are initially expressed inthe neural plate border and then in the NC cell precursors to the formation of cranialnerves The molecular mechanisms of control during embryonic development havebeen gradually elucidated pointing to an important role of transcriptional regulatorswhen neural differentiation occurs However some of these proteins have an importantparticipation in malformations of the cranial portion and their mutation results in aberrantneurogenesis This review aims to give an overview of the role of cell signaling and of thefunction of transcription factors involved in the specification of ganglia precursors andneurogenesis to form the NCderived cranial nerves during anogenesisKeywords cranial nerve peripheral nervous system hindbrain cell signaling transcriptional regulatory networktrigeminal nerve facial nerve vagus nerveAbbreviations BMP bone morphogenetic proteins CN cranial nerve CNS central nervous system DRG dorsal rootganglia FP floor plate hESCs human embryonic stem cells Msx Muscle segmentrelated homeobox NC neural crestNCCs neural crest cells NT neural tube PA pharyngeal arches Pax Paired box PNS peripheral nervous system rrhombomere RA retinoic acid RTK receptor tyrosine kinase Shh Sonic hedgehog Sox Srybox VSMC vascular smoothmuscle cells Wnt Wingless and Int1 WRPW TrpArgProTrp Zic Zinc finger protein of cerebellumEdited byMichael PiperThe University of QueenslandAustraliaReviewed byRoberto MayorUniversity College LondonUnited KingdomRebecca McLennanStowers Institute for MedicalResearch United StatesCorrespondenceManuel J AybarmanuelaybarfbqfunteduarIv¡n Velascoivelascoifcunammx These authors have contributedequally to this workSpecialty sectionThis was submitted toStem Cell Researcha section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted June Published August CitationMndezMaldonado KVegaLpez GA Aybar MJ andVelasco I Neurogenesis FromNeural Crest Cells MolecularMechanisms in the Formationof Cranial Nerves and GangliaFront Cell Dev Biol 103389fcell202000635Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alINTRODUCTIONDuring the embryonic development of vertebrates one of themain events after the gastrulation process is neurulation whichallows the formation of the neural tube NT The neuralectoderm generates not only the central nervous system CNSbut also another set of cells between the NT and the nonneural ectoderm located in the most dorsal part of the NT calledthe neural crest NC Hall SimµesCosta This versatile and plastic cell population was first described byWilhelm His years ago Hall The NC is one of themost important features that separate vertebrates from otherchordate anisms It arises at the posterior and lateral bordersof the neural and nonneural ectoderm the neural plate borderFigure Cerrizuela the embryo NCCs are divided into cranialNC cells NCCs are multipotent and give rise to several celltypes depending on the site of origin along the anteroposterioraxis oftrunkincluding cardiac vagal and sacral A Minouxand Rijli SimµesCosta and Bronner VegaLopez Cranial nerves CN transmit sensory and motorinformation between the brain and tissues of the head andcervical region The CN are formed from the contribution oftwo specialized embryonic cell populations cranial NC andectodermal placodesOrigin of the Neural CrestNCCs which are multipotent delaminate from their origin andmigrate throughout the body to diï¬erentiate into several celltypes including cells of the peripheral nervous system PNSmelanocytes cranial cartilage and bone neuroendocrine cellsand several other phenotypes B In humans at least cell types have been defined as NC derivatives Vickaryousand Hall Proper NC migration relies on environmentalcues such as EphEphrins Smith Semaphorin3F Gammill Versican Szab thechemokine Stromal cellderived factor Theveneau or Robo2 Shiau The migration patternsof NCCs have been clearly described for model anisms likebirds frogs and mice In all vertebrates cranial NCCs emergefrom the forebrain midbrain and hindbrain regions Coulyand Le Douarin Serbedzija Depending ontheir axial origin cranial NCCs will either migrate through thefacial mesenchyme and into the frontonasal process or willpopulate the branchial arches Noden Lumsden Serbedzija The sensory module of the PNSin the cranial region is composed of an array of paired gangliaadjacent to the hindbrain that transduce the perception of touchpain temperature position and special sensory informationfrom the periphery to the CNS Cranial NCCs migrate to formsensory ganglia such as the trigeminal V the facial VII theglossopharyngeal IX the vagus X CN and also to form themotor ganglia for the oculomotor III and accesory XI CNTable and Figures acomplex and multistep processinitially directed by cell signaling molecules including BoneMorphogenetic Proteins BMPs Wnts Wingless and Int1NC formation isNeural CrestDerived Cranial NervesFibroblast Growth Factor FGF and retinoic acid RA Thesesignals reveal the tissue interactions into the ectodermal cellpopulations the neural plate the nonneural ectoderm and theunderlying mesoderm in a highly coordinated manner VegaLopez It has been proposed that NC specificationoccurs during gastrulation as a consequence of the action oftwo successive gradients of secreted signals A combinationof intermediate levels of activity of BMP and Wnt signalingacting on the ectoderm to induce and specify NC precursorsat the neural plate border and a subsequent requirement ofboth signals is needed for maintenance of specification duringneurulation Aybar and Mayor Steventon Inchick embryos it was shown that NCCs are specified as earlyas the blastula stage Prasad It was demonstratedthat during gastrulation Pax7 expression is restricted to cellslocated in a region in the medial epiblast which are NCfated andcontribute to the neural folds and later to migrating NCCs Basch The inhibition of Pax7 function in chicks inhibitedthe expression of key NC markers such as Snai2 OMIM Sox9 Srybox OMIM Sox10 OMIM andHNK1 beta13glucuronyltransferase like OMIM Basch and BronnerFraser This evidence suggests thatthe neural plateprospective ectoderm interaction at the neuralplate border might not be a requisite for NC specification orinduction and that neural plate border formation and NCinduction might be separable eventsThe variousresearch works carried outto study theorigin of NCCs have identified genes anized into a generegulatory network that participate in and control the inductionspecification and diï¬erentiation of NC SimµesCosta An example of this are the transcription factors involvedin induction such as FoxD3 Forkhead Box D3 OMIM Snai2 and Sox9 SaukaSpengler and BronnerFraser Garc­aCastro and coworkers identified a novel preneuralborder state characterized by early Wntβcatenin signalingtargets that displayed diï¬erent responses to BMP and FGFsignaling from the neural border genes in human cells Leung These preborder genes Gbx2 Gastrulation brainhomeobox OMIM SP5 OMIM Zic3 OMIM and Zeb2 OMIM had their induction andpeak of expression before the classical neural plate borderspecifier genes such Msx12 Muscle segmentrelated homeobox OMIM Pax37 OMIM and Zic1 OMIM Such specifier genes together withsignaling molecules direct the expression of NCspecific geneslike AP2 OMIM FoxD3 Snai2 Sox9 and Sox10Specifiers regulate NC eï¬ector genes involved in migrationSox9 Sox10 Cad7 and diï¬erentiation [Col1a Collagen typeI alpha OMIM Ngn1 Neurogenin OMIM MitfMicrophthalmiaassociated transcription factor OMIM] in human NC development Betters The NC population migrates to diï¬erent regions of the mouseembryo from the NT after the epithelialmesenchymal transitionmaintaining its multipotential character until completingdiï¬erentiation in its final destination Baggiolini To study the ontogeny of the NC diï¬erent model anismsboth in vivo and in vitro have been used Several proteinsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Neural crest origin regions in human and mouse embryos and some of its cranial derivatives A The topleft part of the scheme shows the origin of theneural crest cells green that migrate through the embryo On the topright side the level of axial origin see axial color key of different regions of the neural crest isrepresented in developing mouse or equivalent human embryos the migration of neural crest is represented in green inside the embryos and the direction ofmigration is marked with black arrows Depending on their axial level of origin and migratory pathways neural crest cells adopt different fates and contribute tovarious tissues and ans B The main cranial derivatives labeled in green are shown Abbreviations d days E mouse embryonic stage NCCs Neural CrestCells s somite St human stage VSMC vascular smooth muscle cellsincluding transcription factors as well as epigenetic modifiersthat take part in the specification and diï¬erentiation of the NChave been described The study of transcription factors and ofthe signaling pathways in which they participate is importantto understand the diï¬erentiation programs and how thesemultipotent cells are committed to a specific destination On theother hand transcriptome analysis during the development of theNC from specification to migration Meulemans and BronnerFraser and a more recent study covering the migrationto the diï¬erentiation of the NC show the importance of theinteraction between the diï¬erent transcription factors and thesignaling pathways at every stage of NC development SimµesCosta However these authors acknowledge thatit is difficult to have a complete global map since only a fewtranscriptional regulators have been characterized and little isknown about the function of the products of the eï¬ector genesacting on NC migration Betancur SimµesCosta andBronner VegaLopez NC and cranial placodes are thought to appear togetherduring the evolution of vertebrates to give rise to specific sensorystructures of the head Northcutt and Gans Northcutt The components of the sensory nervous system of the headare derived from the NC and from an embryonic cell populationdeveloping in close proximity the cranial sensory placodes theolfactory lens otic trigeminal epibranchial and paratympanicplacodes A series of events induce develop and anizethese cell precursors which through reciprocalinteractionswith NCCs build the functional sensory system in vertebratesSteventon Singh and Groves Migrating NCCsarrive first at the site of ganglia development ie the trigeminalFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cFrontiersinCellandDeveopmentallliBoogywwwfrontiersniAugustlVoumeAlrticeTABLE Contributions of neural crest cells and placodes to ganglia and cranial nervesCranial nerveGanglion and typeOrigin of neuronsReferencesCNI “ Olfactory Ensheating gliaof Olfactory nervesCNIII “ Oculomotor mCiliary visceral efferentCNV “ Trigeminal mixTrigeminal general afferentCNVII “ Facial mixSuperior general and special afferentCNVIII “ Vestibulocochlear sCNIX “ Glossopharyngeal mixCNX “ Vagus mix Superiorlaryngeal branch and recurrentlaryngeal branchCNXI “ Accessory mInferior geniculate general and special afferentSphenopalatine visceral efferentSubmandibular visceral efferentAcoustic cochlear special afferent and Vestibularspecial afferentSuperior general and special afferentInferior petrosal general and special afferentOtic visceral efferentSuperior general afferentInferior nodose general and special afferentVagal parasympathetic visceral efferentNo ganglionTelencephalonolfactory placode NCCs at forebrainNCCs at forebrainmidbrain junction caudal diencephalonand the anterior mesencephalonNCCs at forebrainmidbrain junction from r2 into 1st PAtrigeminal placodeHindbrain NCCs from r4 into 2nd PA 1st epibranchialplacode1st epibranchial placode geniculateHindbrain NCCs 2nd PAHindbrain NCCs 2nd PAOtic placode and hindbrain from r4 NCCsHindbrain NCCs from r6 into 3rd PA2nd epibranchial placode petrosalHindbrain NCCs from r6 into 3rd PAHindbrain NCCs from r7r8 to 4th and 6th PAHindbrain NCCs 4th and 6th PA 3rd nodose and 4thepibranchial placodesHindbrain NCCs 4th and 6th PAHindbrain from r7r8 to PA NCCs 4th PABoyd Muller O™Rahilly andM¼ller Barraud Noden Couly Wahl Lee d™AmicoMartel and Noden Forbes and Welt D™amicoMartel and Noden D™amicoMartel and Noden Lumsden Barlow and Northcutt Begbie andGraham Barlow Krimm Sandell Narayanan and Narayanan D™amicoMarteland Noden O™Rahilly and M¼ller Barlowand Northcutt Narayanan and Narayanan D™amicoMarteland Noden Muller and O™Rahilly O™Rahilly and M¼ller Abbreviations CN Cranial Nerve m purely motor nerve mix mixed nerve sensory and motor NC neural crest PA pharyngeal branchial arch r rhombomere s purely sensory nerve There is no known ganglionof the accessory nerve The cranial part of the accessory nerve sends occasional branches to the superior ganglion of the vagus nerveMndezMadonadoletalNeuralCrestDerivedCranailNerves\x0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Contribution of neural crest cells to the formation of cranial nerves I III V VII VIII IX X and XI These selected cranial nerves are formed by thecontribution of cranial placodes and neural crest cells indicated in green Neural crestderived Schwann cells produce peripheral myelination of cranial nerves III“XIIThe sensory nerves are the olfactory I the optic II and the vestibulocochlear VIII The motor nerves are the oculomotor III the trochlear IV the abducens VIand the accessory XI The remaining are mixed nervesganglion but the diï¬erentiation of these cells is delayed untilthe migration and diï¬erentiation of the corresponding placodalcells in chicks Covell and Noden Placodal specificationand development as well as its contribution to the assembly ofplacodal derivatives is a complex and wideranging topic thatis beyond the scope of this review We will focus on discussingthe main signaling pathways and relevant transcription factorsinvolved in the specification of cranial NCCs precursors theirdiï¬erentiation to form CNs and ganglia that are exclusively NCderived and the alterations caused by the mutations of certaingenes that are important for the neurogenesis of NC derivativesSIGNALING PATHWAYS INVOLVED INCRANIAL NEURAL CRESTDEVELOPMENTareseveralsignaling pathwaysThereand transcriptionfactors that are known to regulate NC and CN formationduring development We discuss some important pathwaysinvolved in cranial NCCs induction and specification in closerelationship with the cranial ganglia and nerves derived from theNC Figure BMPsBone morphogenetic proteins are proteins that control severalimportant steps in the formation and diï¬erentiation of the CNSof vertebrates These proteins act in diï¬erent regions of theCNS to regulate fate proliferation and diï¬erentiation Aftergastrulation the presence of BMPs and the activation of thissignaling pathway are essential for the diï¬erentiation of thenonneural ectoderm whereas the inhibition of this pathway isrequired for the proper formation of the neural plate It has beenproposed that the later activation of BMPs receptors participatesin the induction of the NC through a very fine regulation wherethe presence of BMPs at a specific time will give rise to the NCin mouse and human Embryonic Stem Cells ESCs Figure 3BMizuseki Leung Seminal studies in Xenopus have shown that there is an activitygradient of BMPs controlled by their antagonists and that anintermediate level is needed to induce the formation of the NCLaBonne and BronnerFraser Marchant Barth Tribulo Thus the BMP antagonistsChordin OMIM and Noggin OMIM areexpressed in a spatiotemporal manner thatinfluences theformation of the NC In mouse at embryonic day E Nogginis expressed in the neural folds and in the dorsal region afterthe closure of the NT The expression of Chordin is low atthe level of the neural plate and in the paraxial mesodermThese antagonists participate in the induction of NC as wellas in delamination but also protect from apoptosis induced byBMP during migration and diï¬erentiation of NCCs Importantlyit was observed that the decrease in the expression of theseBMP antagonists alters the PNS derived from the NC andcraniofacial skeletal elements Noggin knockout mice presentedall cranial nerves but the vagus X and glossopharyngeal IX aredisanized and fused Doubleknockout mice of Noggin andChordin lack CN and only a structure similar to the trigeminalganglion V is present Anderson In the chickembryo the activity of BMP signaling during the formation ofNC precursors is modulated by CKIPSmurf factors throughthe regulation of Smad degradation resulting in intermediateFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Gene regulatory network involved in neural crest contribution to the formation of cranial nerves The cranial ganglia and cranial nerves are formed inprecise positions along the dorsoventral and anteroposterior axes of the midbrainhindbrain region A The drawing represents a human embryo at stage days somites equivalent to mouse day E9510 somites and chick stage h somites The cell signaling pathways that providedevelopmental cues to neural crest precursors are colorcoded when these factors diffuse the target regions are indicated with arrows with the same color In panelB an idealized scheme of the hindbrain shows the cell signaling gradients and the genes that establish the dorsoventral pattern C The illustration of the human days stage and chick stage hindbrain rendered flat to eliminate cerebral flexures The levels of origin of the neural crest cells NCCs and placodeswhich contribute to the formation on cranial nerves are indicated on the left NCCs from the corresponding rhombomeres also populate other embryo structures in asegmental fashion and generate different craniofacial derivatives The positions of the cranial ganglia and the otic vesicles are indicated on the right side thecontribution of NCCs is indicated in green The segmental nested expression of HOX genes is colorcoded On the right signaling pathways and the expression oftranscription factors involved in cranial nerve CN formation are indicated Adapted from Lumsden and Keynes Noden Yamamoto and Schwarting BallyCuif and Wassef Takahashi and Osumi and M¼ller and O™Rahilly Abbreviations CN cranial nerve FP floor plate Mmesencephalon NCCs neural crest cells OV otic vesicle r rhombomere PA pharyngeal archeslevels of BMP activity required for proper NC formationPiacentino and Bronner In contrast placode progenitorshave diï¬erential BMP signaling requirements as they can bespecified under low or no BMP signaling Thiery A study of human ESCs hESC showed that if BMPs areblocked with Noggin for h on days or of thediï¬erentiation protocol there is a dramatic decrease in theinduction of human NCCs However if the inhibition is madeon day the inhibition is partial so the participation of BMPsat the beginning of the induction of the NC is very importantwhile the inhibition of this pathway promoted the expressionof neural genes such as SOX1 OMIM HES5 OMIMFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial Nerves and SOX2 OMIM Leung Thisprotocol produced sensory peripheral neurons and it will beof interest to investigate if such neurons can contribute to thesensory CN after grafting them in experimental animals aswell as the eï¬ect of modulating BMPs on peripheral neurondiï¬erentiation Interestingly BMP antagonism upregulates theseneural stem cell markers but several reports indicated that Sox1Hes5 and Sox2 are involved in the suppression of neuronaldiï¬erentiation by maintaining neural stem and progenitorcells in an undiï¬erentiated state in mammalian cells Kan BaniYaghoub The generation ofneurons from stem cells depends on the decrease of Sox13expression caused by proneural proteins However if Sox13target genes were repressed independently of proneural activityneural progenitor cells diï¬erentiated prematurely and someneuronal features emerged These results demonstrate a dualrole of proneural proteins in the acquisition of a definitiveneuronal fate and indicate that the proneural proteindirectedrepression of Sox13 expression is a required and irreversiblestep in the commitment to neuronal diï¬erentiation in severalspecies including mammals Guillemot Farah Bylund BMP4 OMIM and Smad proteins have beeninvolved in an interesting mechanism called retrograde signalingin trigeminal ganglia from rats Ji and Jaï¬rey Thismechanism elicits a specific transcriptionalresponse thatcontributes to the specification of diï¬erent subpopulations ofsensory neurons in the trigeminal ganglia CN V As axonsfrom the neurons oftrigeminal ganglia grow and extendinto their three main peripheral axonal branches ophthalmicmaxillary and mandibular that innervate the correspondingregions of the face they encounter BMP4 which results in aretrograde signal that leads to transport back transcription factorsSMAD1 and from axons to the somata where nuclearaccumulation of the phosphorylated and transcriptionally activeSmad forms contributes to neuronal specification and gangliapatterning Nohe Ji and Jaï¬rey BDNF BrainDerived Neurotrophic Factor OMIM signaling wasalso found to regulate axonal levels of SMAD1 and inconcert with BMP4 for patterning of the trigeminal gangliaJi and Jaï¬rey Hippo PathwayGenetic studies have demonstrated that Hippo signaling is crucialin an size regulation controlling cell number by modulatingcell proliferation and apoptosis processes Huang Hippo is a criticalfactor for proliferation and epithelialmesenchymal transition during embryonic development andcancer In the neural tube of the mouse chicken and frogYAP YesAssociated Protein OMIM is expressed inthe ventricular zone progenitor cells and colocalizes with theneural progenitor cell marker Sox2 Milewski Cao It has been observed that the ectopic expressionof one ofthis pathwayTAZ Transcriptional Coactivator With PDZBinding MotifOMIM in mammalian cells stimulates cell proliferationthe transcriptional regulators ofreduces the inhibition by contact and promotes the epithelialmesenchymal transition Lei A relationship between this signaling pathway and the classicalNC genes such as interaction with Pax3 is through TAZ andthe phosphoprotein YAP65 These proteins participate as coactivators of Pax3 It has been suggested using transgenicmice that Tead2 TEA Domain Family Member OMIM is an endogenous activator of Pax3 in NCCs Milewski Through expression assays Pax3 and Yap65were colocalized in the nucleus of NC progenitors in thedorsal region offorSchwann cell proliferation and diï¬erentiation in a stagedependent manner Nuclear TAZYAP complexes activate cellcycle regulators to promote Schwann cell proliferation whiledirecting diï¬erentiation regulators in cooperation with Sox10 formyelination in rodents Deng the NT HippoTAZYAP are criticalNeurofibromatosis Nf2 OMIM is a tumorsuppressor that inhibits YAP during dorsal root ganglia DRGdevelopment Merlin encoded by the NF2 tumorsuppressivegene was identified through genetic studies in mouse embryosand proved to be an important upstream regulator of theHippoYap pathway Neurofibromatosis is an inherited diseasecharacterized by the development of bilateral Schwann celltumors originated from CN VIII Mouse with specific Schwanncellinactivated Nf2 alleles developed schwannomas and SChyperplasia McClatchey Giovannini Merlin has also been shown to act as a suppressor ofmouse neural progenitor proliferation by inhibiting TAZYAPpathway activity Lavado The mechanism bywhich Merlin regulates YAP activity might involve p21 Proteinactivated kinase PAK1 OMIM activation whichinduces phosphorylation of Merlin thus abrogating its scaï¬oldfunction for YAP and LATS12 OMIM andthereby attenuates YAP phosphorylation by LATS12 in mousecells Sabra it has been suggested that nuclearexport signals of Merlin mediate YAP nuclear export in epithelialmammalian cells Furukawa Hindley and coworkers investigated the role of HippoYAPsignaling in NC development and neural diï¬erentiation Theyshowed thatthe activity of YAP promotes an early NCphenotype accompanied by premature migratory behavior andthat HippoYAP interacts with RA signaling in hESCs Hindley A recent study demonstrates that YAP is necessaryfor the migration of a premigratory pool of NCCs sincethey incorporated YAP signaling into a BMPWntdependentmolecular network responsible for the migration of trunklevelNC in avians Kumar Notch SignalingNotch is a family of conserved receptors whose activation isinduced by specific ligands Delta1 OMIM Delta OMIM Delta4 OMIM Jagged1 OMIM and Jagged2 OMIM through interactionwith four possible receptors Notch14 Perdigoto and Bardin Once the Notch receptors are activated through thecellcell interaction proteolytic cuts are carried out resultingin the release ofthe Notch Intracellular Domain NICDFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesMumm NICD translocate to the nucleus andforms a transcriptional complex together with the DNA bindingprotein CBF1 C promoter binding factor OMIM This complex recognizes the specific sequence CTGTGGGAAin itstarget genesfor example Hes1 OMIM Kageyama Notch1 receptor is present during development oftherhomboencephalon at E95 in mice showing strong expressionwithin the hindbrainincluding the trigeminal geniculatepetrosum and nodose placodes which give rise to CN VVII IX and X respectively and is also expressed in the oticand olfactory vesicle Reaume A study wherehuman induced pluripotent stem cells were induced toward NCdiï¬erentiation showed that when Notch signaling is blockedusing a Îsecretase inhibitor DAPT or shRNA for JAGGED the genes specifying NC [DLX5 Distalless homeobox OMIM PAX3 SNAI2 SOX10 and TWIST1 OMIM] are downregulated However the ectopic expression ofNICD1 increased its expression demonstrating that Notch alsoparticipates significantly in NC induction Noisa Mead and Yutzey evaluated the function of Notch signaling inmurine NCderived cell lineages in vivo They demonstratedthat cellautonomous Notch has an essential role in properNCCs migration proliferation and diï¬erentiation with criticalimplications in craniofacial cardiac and neurogenic developmentMead and Yutzey Sonic HedgehogSonic Hedgehog Shh signaling is involved in the correctdevelopment of NC and therefore in the generation of its cellularderivatives Figure 3B Shh is a member of the family ofthe secreted Hedgehog proteins Sonic Shh OMIM Indian Ihh OMIM and Desert Hedgehog Dhh OMIM Shh regulation during NC diï¬erentiation is crucialduring head and face morphogenesis Mutant mice and humanslacking Shh present holoprosencephaly and cyclopia due to thelack of separation of the forebrain lobes Chiang It is suggested that Shh inhibition maintains Pax3 expressionso the lack of Shhmediated regulation for Pax3 inductionpromotes the constitutive induction of NC generating theaforementioned phenotypes A subset of Fox genes regulated byShh signaling is important during lip morphogenesis in miceEither Shh addition or Foxf2 OMIM overexpressionwas shown to be sufficient to induce cranial NCCs proliferationEverson On the other hand enhanced Shh signaling in mousemediated by lossoffunction Ptch1WigWig of the Shh receptorPatched1 Ptch1 OMIM suppressed canonical Wntsignaling in the CN region This critically aï¬ected the survivaland migration of cranial NCCs and the development ofplacodes as well as the integration between NC and placodesKurosaka Ptch1WigWig mutants exhibited severelydisanized trigeminal CNV and facial nerves CNVII thatdid not develop properly and failed to project to their appropriatetargettissues Kurosaka High levels of Shhsignaling have been correlated with Moebius Syndrome whichis characterized by cranial nerve defects including trigeminalabducens CNVI and facial alterations concurrent with othercraniofacial defects Verzijl VegaLopez NCCs migration is particularly sensitive to Shh levels since inmice lacking Shh these cells continue their migration beyondthe normal position and fuse medially condensing into a singlemidline ganglion Fedtsova Mutation in the mouseHedgehog acyltransferase Hhat OMIM gene producedhypoplasia and aberrant fusion of cranial ganglia CN V VII IXand X and aï¬ected NC and placode gene markers expressionsuggesting that a regionalized action of the Hedgehog signalingis required for proper cranial ganglia and nerve developmentand patterning Dennis In vitro analyses showedthat Shh increased the number of cranial NC progenitors fromquail embryos yielding neural and mesenchymal lineages Shhcan decrease the neuralrestricted precursors without aï¬ectingsurvival or proliferation These data also suggestthemesenchymalneural precursor was able to yield both the PNSand superficial skeleton Calloni thatReceptor Tyrosine Kinase RTK FamilyHumans have known RTKs which fall into subfamiliesA few years ago a systematic work summarized the contributionof the mouse model to the understanding of the role of a subsetof RTKs in regulating the activity of NCCs in developmentFantauzzo and Soriano With respect to its downstreamsignaling RTKs induce the activation of various pathwaysincluding PLCÎ PI3K MAPK JNK Shc Erk and the JAKSTATpathways In this section we discuss insights pointing tomechanisms of action of some RTK families in relation tothe development of the cranial NC that have emerged fromrecent evidenceEph ReceptorsEphrin ligands and Eph erythropoietinproducing humanhepatocellular carcinoma receptors comprise an increasinglywell studied family of signaling molecules Ephrins bind totwo families of transmembrane tyrosine kinase receptors EphAand EphB While Atype Ephrins preferentially bind to EphAreceptors Btype Ephrins do so to EphB receptors In Xenopusthe streams of NCCs going to the second branchial arch expressEphrinB2 whereas cells reaching the third arch express EphB1disruption of EphEphrin signaling results in aberrant migrationof NCCs causing mixing of the streams in the branchial pouchesSmith Eph receptor functions are best characterizedin the mouse nervous system where they are involved inneuronal development and axon guidance Wilkinson Xuand Henkemeyer migration and proliferation Conover H

bladder cancer is the tenth most common cancer globally but existing biomarkers and prognostic models are limitedmethod in this study we used four bladder cancer cohorts from the cancer genome atlas and gene expression omnibus databases to perform univariate cox regression analysis to identify common prognostic genes we used the least absolute shrinkage and selection operator regression to construct a prognostic cox model kaplan“meier analysis receiver operating characteristic curve and univariatemultivariate cox analysis were used to evaluate the prognostic model finally a coexpression network cibersort and estimate algorithm were used to explore the mechanism related to the modelresults a total of genes were identified from the four cohorts to construct the prognostic model including eight risk genes serpine2 prr11 dsel dnm1 comp elovl4 rtkn and mapk12 and three protective genes fabp6 c16orf74 and tnk1 the 11genes model could stratify the risk of patients in all five cohorts and the prognosis was worse in the group with a highrisk score the area under the curve values of the five cohorts in the first year are all greater than furthermore this model™s predictive ability is stronger than that of age gender grade and t stage through the weighted coexpression network analysis the gene module related to the model was found and the key genes in this module were mainly enriched in the tumor microenvironment b cell memory showed low infiltration in highrisk patients furthermore in the case of low b cell memory infiltration and highrisk score the prognosis of the patients was the worst the proposed 11genes model is a promising biomarker for estimating overall survival in bladder cancer this model can be used to stratify the risk of bladder cancer patients which is beneficial to the realization of individualized treatmentkeywords bladder cancer cox regression prognostic model overall survivalcorrespondence yyzhucmueducn yumengcmueducn jiaxing lin and jieping yang contributed equally to this work department of urology the first hospital of china medical university shenyang liaoning china department of reproductive biology and transgenic animal china medical university shenyang liaoning chinafull list of author information is available at the end of the bladder cancer is the tenth most common cancer in the world it is more common in men than in women and the morbidity and mortality rate in men is four times higher than that in women a significant risk factor for bladder cancer is smoking with half of all cases are linked to smoking [ ] about of patients with nonmuscular invasive bladder cancer are treated by radical tumor resection followed by intravesical instillation of the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clin a0et a0al cancer cell int page of bacille calmettegurin vaccine approximately of patients have muscular invasive or metastatic bladder cancer and are treated with radical cystectomy and neoadjuvant chemotherapy bladder cancer is a complex disease although many clinical factors and molecular markers have been identified that can predict prognosis these have low accuracy and it does not have universal applicabilitywith the continued development of gene sequencing technology and expansion of public databases it is possible to take advantage of biological information to mine sequencing data and identify biomarkers this method can utilize large sample sizes with less investment making it an important new direction to screen disease biomarkers of available databases the cancer genome atlas tcga cance rgeno menihgov database is an authoritative oncology database and the gene expression omnibus geo httpwwwncbinlmnihgovgeo database stores curated gene expression datasets many studies have constructed a multiqueue verification model based on these two databases such as nonsmall cell lung cancer [ ] and ovarian cancer prognostic models provide effective guidance for doctors and patients to make optimal treatment decisions however in the study of the bladder cancer model many models can only be verified in two or three cohorts [ ] and do not have clinical extensibilityin this study gene expression and clinical data related to bladder cancer were obtained from tcga and geo databases and common prognostic genes were screened by univariate cox proportional hazard regression this prognostic model of bladder cancer was constructed by least absolute shrinkage and selection operator lasso regression and then verified using five cohorts this robust model can help patients with bladder cancer to achieve individualized treatmentmaterials and a0methodsdata obtaining and a0processingto reduce the error of the data we searched the tcga and geo databases for bladder cancer cohorts with a sample size of more than and these cohorts need to include survival status and survival time we found a total of five cohorts the raw rna sequencing and clinical data of bladder urothelial carcinoma blca n were obtained from tcga database and the raw rna sequencing and clinical data of gse13507 n gse32548 n gse32894 n and gse48075 n from the geo database these five cohorts were analyzed on the illumina sequencing platform in r programming language software the r package œedger was used to standardize the raw rna expression matrix and obtain the corresponding log valuesconstruction of a0prognostic modelthe cox proportional hazard regression model was applied to perform univariate cox proportional hazard analysis of all genes in tcgablca gse13507 gse32548 and gse32894 cohorts the hazard ratio hr from univariate cox regression analysis was used to select the genes that were positively or negatively related to prognosis a gene with hr was considered a risk gene and a gene with hr was considered a protective gene statistical significance was defined as p the genes with hr and p were selected from the four cohorts and then risk genes were obtained by overlapping four groups of genes similarly genes with hr and p were selected for the four cohorts and combined to obtain the set of protective genes a venn diagram was constructed using the online tool bioinformatics and evolutionary genomics httpbioin forma ticspsbugent bewebto olsvenn the identified risk and protective genes make up a set of prognostic genesthe data from tcgablca as a training set was used to construct a prognostic model to simplify the model the genes were selected by univariate cox regression analysis with a p value less than the r package œglmnet and œsurvival were used to do lasso regression to further screen genes and construct a cox module first the function œglmnet was randomly simulated times to construct the model and establish the relationship between lambda punishment coefficient and regression coefficients coef a higher value of lambda corresponds to greater punishment with the increase of lambda some gene coef become zero indicating that the expression of the gene will not affect the model so this gene can be removed from the model then the function œcvglmnet was randomly simulated times for crossvalidation cv cv is usually divided into holdout kfold and leaveoneout cv the function used kfold cv and k took the default parameter in tenfold cross validation the data set is divided into equal parts and then nine part are tested as training sets and one is used as the validation set the deviance of the tests were used to estimate the accuracy of the model when the deviance is minimum the model is the best and the coef of the model can then be obtained by using the corresponding lambda value finally we obtained the genes and the corresponding coef to build the model the prognostic model was defined as risk score ˆ‘ni expi · coefi where n is the number of genes expi is the expression of the ith gene and coefi is the regression coefficient of the ith gene the algorithm can prevent overfitting of the model remove highly coexpressed 0clin a0et a0al cancer cell int page of genes and finally construct a simplified model using the obtained model we calculated the risk score of each patient in the four cohortsbetween genes modulemembership and the correlation between genes and clinical traits genesignificance kaplan“meier analysisr packages œsurvival and œsurvminer were used for kaplan“meier analysis and the function œrescat was used to find the best cutoff value of factors the cutoff was used to divide the sample into a highrisk group and a lowrisk group to construct the kaplan“meier curve with the smallest p value the risk score distribution gene expression and patient survival status data were plotted using the r package œpheatmapreceiver operating characteristic curvereceiver operating characteristic roc curves of a0 years were plotted and the area under the curve auc values were calculated using the r package œsurvivalrocunivariate and a0multivariate cox regression analysisthe risk scores and clinicopathological factors were analyzed by univariate and multivariate cox regression analysis using the r package œsurvival the multivariate cox analysis included age sex primary tumor range t stage grade and risk score tcgablca and gse13507 also include stage lymph node and metastasisexploration of a0gene methylationœcbioportal for cancer genomics is an openaccess opensource resource wwwcbiop ortal for interactive exploration of multiple cancer genomics data sets [ ] use this tool to query the relationship between gene expression and dna methylation in the œbladder cancer tcga cell  dataset the tool can also download gene methylation data which can be combined with clinical data for kaplan“meier analysisweighted co‘expression network analysisthe set of mrna genes in the tcgablca cohort with univariate cox analysis values less than were selected to construct a bladder cancer coexpression network by weighted gene coexpression network analysis wgcna the r package œwgcna was used to construct the coexpression network this method takes advantage of similarities of gene expression and groups the genes with similar expression patterns into the same module with the idea that genes in the same module may share physiological function we then explored the relationship between the clinicalfactorriskscore and module and applied pearson correlation to determine the module that was most related to the risk score the key genes were selected by the calculated correlation pathway and a0process enrichment analysisidentified genes were entered into the metascape database httpmetas cape for pathway and process enrichment analysis the enrichment analysis included œkegg pathway go biological processes reactome gene sets canonical pathways and corum to evaluate the potential biological functions and pathways of the selected genescibersort and a0estimate algorithmcibersort celltype identification by estimating relative subsets of rna transcripts is a bioinformatics algorithm to calculate cell composition from gene expression profiles of complex tissues the combination of cibersort and lm22 leukocyte signature matrix can be used to calculate the content of kinds of human leukocyte subsets we used the r package œcibersort to calculate the number of immune cells in each sample of the tcgablca cohort estimate estimation of sttromal and immune cells in malignant tumours using expression data is a tool that uses gene expression trends to infer the fraction of stromal and immune cells in tumor samples the immune score of each patient in tcgablca was calculated by the r package œestimate immune score represents the content of immune cells and the higher the score the higher the cell contentstatistical analysisall the statistical analyses were carried out by using r programming language software rx64 all r packages were obtained from cran cranrproje ct or bioconductor httpwwwbioco nduct or the two groups were compared by the wilcoxon test and comparison between multiple groups was performed by kruskal“wallis test statistical significance was defined as p difference scatter plots were constructed using the r package œbeeswarm we used the r package œvioplot to draw violin pictures and the r package œcorrplot to draw correlation heat mapresultsdata processing and a0research processwe obtained the raw rna sequencing and clinical data of tcgablca n gse13507 n gse32548 n and gse32894 n we utilized data only from patients associated with rna sequencing data survival time survival status and primary tumor for further analysis the basic clinical information of the remaining patients is summarized in table a0 the sample 0clin a0et a0al cancer cell int page of table basic clinical information for a0the a0four cohortsclinical factorstcga_blcan age ‰¦ gender male femalet stage t2 ‰§ t2grade who2004 low highgrade who1999 g1 g2 g3vital status alive deadfollowup mean ± sd year ± ““““““gse13507n “““gse32548n “““““““gse32894n ““ ± ± ± ““sd standard deviationsizes of these four cohorts are all greater than the grade of bladder cancer is closely related to recurrence and invasive behavior two grading methods were used in these four cohorts tcgablca and gse13507 used the who grading standard of which was divided into punlmp papillary urothelial neoplasms of low malignant potential low grade and high grade gse32548 and gse32894 used the who grading standard of which was divided into grade g1 grade g2 and grade g3 the research process is shown in fig a0construction of a0prognostic modelunivariate cox proportional hazard analysis was carried out in tcgablca gse13507 gse32548 and gse32894 cohorts there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p combining the four datasets allowed identification of risk genes and protective genes fig a0 because of the large sample size of tcgablca we used this cohort to build the prognostic model first genes with univariate cox pvalues less than in tcgablca were selected then the genes were analyzed by lasso regression analysis fig a0 2a when the number of genes in the model was the deviance was the smallest fig a02b according to the lambda value the corresponding coef of the selected genes could be determined the prognostic model could then be constructed by using the corresponding coef of the genes to see more intuitively whether these genes are collinear we analyze the coexpression of these genes as shown in the fig a02c the coexpression index of none of these two genes is greater than finally we successfully constructed a prognostic module risk score serpine2 prr11 fab p6 ˆ’\u200b c 16orf74 ˆ’\u200b ds el d nm1 \u200b comp t nk1 ˆ’\u200b elov l4 rtkn mapk12 the basic information and coef values of the genes are listed in additional file a0 table a0 s1 the average expression values transcripts per million of genes in the four cohorts are greater than which is of practical significance for detection additional file a0 table a0s2 the results of univariate regression analysis of these genes in cohorts are shown in additional file a0 table a0s3 0clin a0et a0al cancer cell int page of fig flow chart of analysiskaplan“meier analysis of a0 geneseleven genes were taken kaplan“meier analysis in cohorts using the heat map to show the results of the study fig a0 2d except for dsel in gse32894 and c16orf74 in gse32548 the other analyses were statistically significant p serpine2 rtkn prr11 mapk12 elovl4 dsel dnm1 and comp showed that the prognosis of patients with high expression was worse and the analysis of elovl4 in tcgablca was taken as an example p fig a0 2e tnk1 fabp6 and c16orf74 showed that the prognosis of the low expression group was worse and the analysis of fabp6 in tcgablca was taken as an example p fig a02fthe degree of a0dna methylation of a0tnk1 and a0c16orf74 was a0negatively correlated with a0gene expressiondna methylation can regulate gene expression we explored the relationship between expression and methylation of these genes additional file a0 figure s1a“k the results showed that there was a negative correlation between tnk1 gene methylation and gene expression spearmen cor ˆ’ p 144eˆ’ so did as c16orf74 spearmen cor ˆ’ p 897eˆ’ then we took tnk1 and c16orf74 methylation data combined with clinical data for kaplan“meier analysis we found that the degree of methylation of these two genes can predict the prognosis of bladder cancer p additional file a0 figure s1i m and the prognosis is worse in the case of hypermethylation the expression of tnk1 and c16orf74 is inhibited by hypermethylation which leads to a worse prognosis of bladder cancerverification of a0the a0prognostic modelthe prognostic model was used to calculate the risk scores of each patient in the training set tcgablca and three test sets gse13507 gse32548 and gse32894 we identified the best cutoff value with a risk score of for tcgablca using this method the cutoff values of gse13507gse32548gse32894 were the kaplan“meier curves showed that the prognosis of patients with highrisk was significantly worse than that of patients with lowrisk in the four cohorts p fig a03a“d the receiver operating characteristic roc curves of the four cohorts were drawn the a0year area under the curve auc values for the tcgablca cohort were and respectively fig a0 3e those for the gse13507 cohort were and respectively fig a03f those for the gse32548 cohort were and respectively fig a03g and those for the gse32894 group were and fig a03h additional file a0 figure s2 shows the risk score distribution gene expression values and survival status of patients in both the highrisk group and the lowrisk groupthe clinical factors and risk scores of the four cohorts were analyzed by univariate cox and multivariate cox regression analysis table a0 the results of univariate analysis showed that t stage was more effective in predicting prognosis among the clinical factors 0clin a0et a0al cancer cell int page of fig lasso cox and kaplan“meier analysis a lines of different colors represent different genes with the increase of lambda value the coef of some genes become zero indicating that they do not affect the model b the deviance of the cross validation when the partial likelihood deviance is minimum the corresponding model is the best c the coexpression heat map of genes red indicates a positive correlation blue indicates a negative correlation and the cross indicates no statistical significance d the heatmap of kaplan“meier analysis results of genes red means high expression of the gene lead to worse prognosis blue means low expression of the gene lead to worse prognosis grey means there are no significance of the analysis p means it is statistically significant the darker of the color shows the smaller of the pvalue e kaplan“meier analysis of elovl4 in tcgablca f kaplan“meier analysis of fabp6 in tcgablcaand three cohorts had statistical significance the risk scores were statistically significant in all four cohorts and the p value of three cohorts was lower than that of the t stage in multivariate cox analysis risk scores were statistically significant in three cohorts indicating that the three cohorts were independent of other clinical factors in predicting prognosis in this analysis only two cohorts of t stage had statistical significance so it is obvious that t stage is not as strong as risk score to predict the prognosis finally we compared the risk scores for different grades and t stage in the four cohorts and found that the risk scores increased 0clin a0et a0al cancer cell int page of fig the kaplan“meier analysis and roc curves of the risk score kaplan“meier curves of tcgablca a gse13507 b gse32548 c and gse32894 d red indicates highrisk group and blue indicates lowrisk group p means it is statistically significant ci confidence interval roc curves of tcgablca e gse13507 f gse32548 g and gse32894 h in years and their corresponding auc valueswith the increase of grade and t stage p additional file a0 figure s3a b in the gse32548 cohort we compared the risk scores of fgfr3 and tp53 or with the mdm2 alteration for wild type and mutant type additional file a0 figure s3c shows that a lower risk score of mutant type than that of wild type for the fgfr3 groups p in tp53 or with the mdm2 alteration the score of mutant type was higher than that of wild type p additional file a0 figure s3cseven genes and a0model were successfully verified in a0gse48705we evaluated the prognostic ability of genes and models in gse48075 n the results showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp were statistically significant p additional file a0 figure s4 and the prognosis was worse in the high expression group which was consistent with the analysis result of the previous four cohorts the risk score of patients was calculated according to the model 0clin a0et a0al cancer cell int page of table univariate and a0 multivariate cox regression analysis of a0 clinicalfactorsriskscore with a0 overall survival rate in a0patientsvariablesunivariate analysismultivariate analysishr ciphr ciptcgablca age gender grade stage t stage node metastasis risk scoregse13507 age gender grade stage t stage node metastasis risk scoregse32548 age gender grade t stage risk scoregse32894 age gender grade t stage risk score0inf “ 120eˆ’ 556eˆ’ 991eˆ’ 262eˆ’ 657eˆ’ 806eˆ’ 544eˆ’ 246eˆ’0inf “ 269eˆ’ “ 214eˆ’ 996eˆ’ “ 763eˆ’ “ 223eˆ’ 164eˆ’ 824eˆ’ 367eˆ’ “ 453eˆ’ “ 129eˆ’ “ 246eˆ’ “ 400eˆ’ “ 506eˆ’ “ 388eˆ’ “ 364eˆ’ “ 114eˆ’ “ 934eˆ’ “ 613eˆ’ “ 326eˆ’ “ 742eˆ’ “ 637eˆ’ “ 871eˆ’ “ 181eˆ’ “ 997eˆ’ “ 870eˆ’ “ 183eˆ’ “ 271eˆ’ “ 221eˆ’ “ 737eˆ’ “ 200eˆ’ “ 427eˆ’ “ 183eˆ’ “ 341eˆ’446eˆ’ “ “ 179eˆ’ “ 445eˆ’ “ 445eˆ’ “ 942eˆ’ “154eˆ’ “ 163eˆ’ “ 561eˆ’ “ 408eˆ’ “ 986eˆ’ “136eˆ’italic font means statistically significanthr hazard ratio ci confidence interval inf infinityand the risk score was analyzed by kaplan“meier analysis the prognosis of the high riskscore group was worse and the difference was statistically significant p fig a04a we drew the roc curves of the risk score and the auc value of a0 year was fig a0 4b figure a0 4c showed the risk score distribution gene expression values and survival status of patients between high and lowrisk groupsweighted co‘expression network analysis and a0enrichment analysisthe coexpression network was constructed with coding genes and samples in tcgablca cohort first the expression matrix was transformed into a topological overlap matrix according to β then the genes were divided into different modules fig a05a using the dynamic pruning tree method next the association analysis of clinical traits and modules fig a05b showed a high correlation between the turquoise module and risk score cor p 2eˆ’ there was also a high correlation between the turquoise module and survival status cor p 9eˆ’07grade cor p 1eˆ’09stage cor p 1eˆ’ we selected key genes fig a0 5c in the turquoise module according to the standard to explore the potential function of these key genes pathway and process enrichment analysis of these key genes were performed as shown in fig a05d the three most highly significantly enriched terms were extracellular matrix anization collagen fibril anization and ecm proteoglycans all related to the tumor microenvironment tmeimmune cells can be combined with a0risk scores for a0prognostic analysiswe used cibersort to calculate the infiltration ratio of immune cells in tcgablca samples and used a bar chart to show the infiltration of high and lowrisk groups fig a06a then the wilcoxon test was used to compare the difference between high and lowrisk groups the results showed that b cells naive macrophages m0 and macrophages m1 showed high infiltration in the highrisk group b cells memory dendritic cells resting and dendritic cells activated showed high infiltration in the lowrisk group p fig a06b furthermore we took the risk score and the infiltration degree of these six kinds of immune cells for joint prognostic analysis the samples were divided into four clusters for kaplan“meier analysis according to the median value of the risk score and immune cell infiltration degree the results showed that these groups could also be used for prognostic analysis p fig a06c“h among them the prognostic ability of b cells memory is the best when the degree of b cells memory infiltration is low and the risk score is high the prognosis of this cluster is significantly worse than that of other clusters we used estimate to calculate the tcgablca cohort™s immune score and then combined with the risk score for kaplan“meier analysis the results showed that the cluster with low immunescore and high riskscore had the worst prognosis additional file a0 figure s5 0clin a0et a0al cancer cell int page of fig validation of the model in gse48075 a kaplan“meier analysis of the riskscore b roc curves of riskscore in years c the risk score analysis from top to bottom patient™s risk distribution gene expression profile and survival status mapdiscussionbladder cancer is a heterogeneous disease with a high incidence and recurrence rate but there is no robust predictive tool to guide clinical treatment some recent studies have also constructed a new model for bladder cancer such as dna methylationdriven genes related model and immune genes related model these models prefer to take a kind of gene set to build the model rather than the whole genome into the screening in this study prognostic genes were screened from four cohorts with the whole transcriptome and the common prognostic genes were selected to construct the model the model successfully predicted the overall survival of five cohorts about bladder cancer patients and it is the research with the largest cohort size in the same type of researcha variety of regional source cohorts are used to jointly develop the model which makes the model have higher credibility and broader applicability in our study all genes in all cohorts were then analyzed by univariate cox proportional hazard analysis to screen common prognostic genes in four cohorts after further screening a prognostic model was constructed using the data from the tcgablca cohort instead of using the genes obtained by analysis of a single cohort to construct a prognostic model the prognostic genes common to multiple cohorts were used to make the model more stable and reliable the patients in the tcgablca cohort were from north america gse13507 was from asia and gse32548 and gse32894 were from europe it is concluded that this model has a wide range of applicabilitythe main finding of this study is that the 11gene model we developed has a robust prognostic ability and successfully predicted the prognosis of five cohorts kaplan“meier analysis showed that the prognosis of the highrisk group was worse in all the four cohorts p the 1year auc values of the tcgablca gse13507 gse32548 and gse32894 cohorts were and respectively indicating that the risk score has the ability to predict prognosis univariate and multivariate cox analysis of clinical factors and risk scores showed that the ability of risk scores to 0clin a0et a0al cancer cell int page of fig weighted coexpression network and enrichment analysis a genes were divided into different modules according to the dynamic cutting tree method and different colors represent different modules b the heatmap of the correlation between the gene module and clinical traits p indicates statistical significance c gene significance and module membership scatter diagrams of the turquoise module the dots in the red box are represented as key genes d the left side of the picture shows the interaction network map of enriched proteins and the same color indicates the same enrichment the right side of picture shows the enriched terms decreasing from top to bottom by the significance of enrichmentpredict prognosis was better than age gender grade and t stage we also analyzed the relationship between risk score and different clinical status and found increased risk score with the increase of bladder cancer t stage and grade p there are also significant differences in risk scores between wild type and mutant types of different genes we analyzed the gse32548 mutation data and found lower risk score in the group of fgfr3 mutation in contrast in the presence of a tp53 mutation or with mdm2 alteration the risk score was higher according to previous reports mutations in fgfr3 is associated with better prognosis but tp53 mutation or with mdm2 alteration is associated with worse prognosis [ ] these s indirectly verify the prognostic ability of the risk model finally the 11gene model was successfully verified in independent cohort gse48075 the model is verified by four internal cohorts and one external cohort which shows that the model has the potential to be used in the cliniceleven genes are potential prognostic markers and therapeutic targets for bladder cancer these genes have a stable prognostic ability in tcgablca gse13507 gse32548 and gse32894 cohorts and kaplan“meier analysis showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp was successfully verified in gse48075 besides the methylation level of tnk1 and c16orf74 can also predict the prognosis of bladder cancer among them only c16orf74 and 0clin a0et a0al cancer cell int page of fig combined analysis of risk score and immune infiltrating cells a the bar chart of the infiltration of kinds of immune cells in the samples of high and lowrisk groups b the difference of kinds of immune cells between the lowrisk group and the highrisk group was analyzed an

dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face here we reveal yet another connection between craniofacial development and making ribosomes through the protein paired box pax9pax9 functions as an rna polymerase ii transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans we now expand thisfunction of pax9 by demonstrating that pax9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome this function ofpax9 is conserved to the anism xenopus tropicalis an established model for humanribosomopathies depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors this work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentauthor summarywe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis in humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies here we have discovered a new role for the protein pairedbox pax9 in making human ribosomes while pax9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation farleybarnes ki deniz e overton mmkhokha mk baserga sj paired box pax9 the rna polymerase ii transcription factorregulates human ribosome biogenesis andcraniofacial development genet e1008967 101371 pgen1008967editor paul a trainor stowers institute formedical research united statesreceived february accepted june published august copyright farleybarnes this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all rnaseq andrnapii chipseq files are available from the geneexpression omnibus geo database and areaccessible through the geo series accessionnumber gse154764 wwwncbinlmnihgovgeoqueryacccgiaccgse154764 allnumerical data that underlies graphs or summarystatistics is available in the supporting informations4 table genetics 101371 pgen1008967 august genetics 0cfunding this work was supported by the nationalinstitutes of health r01gm115710r01gm122926 and r35gm131687 to sjbr01hd081379 to mkk t32gm007223 to sjb andkif f31de026946 to kif and a pilot grant from theyale cancer center to sjb the funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptcompeting interests the authors have declaredthat no competing interests existpaired box pax9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of pax9 by showing that pax9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome in addition we show that this function is conserved to the model anismxenopus tropicalis this link between pax9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these pax9 mutations in humansintroductionwhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise for example genetic disruption of thetcof1 polr1c or polr1d genes in the ribosomopathy treacher collins syndrome tcsomim results in reduced preribosomal rna prerrna transcription [“] interestingly while these mutations each affect the global process of prerrna transcriptionpatients have specific defects in craniofacial development tcs patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to mdm2the e3 ligase for p53 [ ] this stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of tcstcs is not the only ribosomopathy to affect craniofacial development for example diamond blackfan anemia dba omim is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] however dba patientsoften also have craniofacial anomalies and cleft palate reviewed in additionally theribosomopathy acrofacial dysostosis cincinnati type omim is caused by mutationsin polr1a that inhibit prerrna transcription resulting in craniofacial defects amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentthe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment in humans these proteins are still being defined previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomaldna rdna into the 47s polycistronic prerrna fig 1a the prerrna is further modified and processed to create the mature 18s 58s and 28s rrnas these rrnas are incorporated along with the 5s rrna and ribosomal proteins into the small ssu and large lsusubunits of the ribosome the 47s prerrna is transcribed by rna polymerase i rnapiwhile the 5s rrna is transcribed by rna polymerase iii rnapiii various assembly factorsand the ribosomal proteins are transcribed by rna polymerase ii rnapii in addition torequiring all rna polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimulithe complex development of the face is controlled by a number of proteins including several rnapii transcription factors such as paired box pax9 pax9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development by genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig pax9 is required for human ribosome biogenesis a ribosome biogenesis at a glance the tandemly repeated ribosomal dna rdna istranscribed into the 47s polycistronic preribosomal rna prerrna by rna polymerase i rnapi this 47s prerrna is processed throughmultiple steps to form the mature 18s 58s and 28s rrnas which are incorporated into the small and large subunits of the ribosome along with the5s rrna and ribosomal proteins ribosomes perform cytoplasmic cellular protein synthesis through the translation of mrnas b pax9 depletionreduces nucleolar number from “ to only in mcf10a cells left panel nuclei stained in hoechst are shown in blue nucleoli are shown in pinkand stained with antifibrillarin antibody as in sigfp top was used as a negative control “ nucleolinucleus and siutp4 middle was usedas a positive control nucleolusnucleus sipax9 is shown at the bottom right panel quantitation of the number of nucleoli per nucleus for sigfptop siutp4 middle or sipax9 bottom c pax9 is not required for rnapi transcription in mcf10a cells a dualluciferase reporter assay wasused to quantify luminescence after sirna depletion of pax9 the plasmids are phrdiresluc firefly to report rnapi transcription and arenilla transfection control as in the ratio of firefly to renilla luciferase was normalized to the sint control n sinol11 was used as apositive control data were analyzed by student™s t test using graphpad prism ���� p � d pax9 is required for pre18s rrnaprocessing in mcf10a cells left schematic of prerrna processing steps in human cells intermediates detected by probe p3 are indicated with ablack box below center northern blot with probe p3 a probe for the 7sl rna was used as a loading control intermediates detected by probe p3 areshown to the right of the northern blot negative controls were mock no sirna and sint nontargeting siutp4 was used as a positive control right quantitation by ramp of probe p3 upper and 7sl lower northern blots graph is mean ± sem n data were analyzed by2way anova using graphpad prism ���� p � ��� p � �� p � and � p � ptp indicates the 47s 45s and 43s processingintermediates e pax9 sirna depletion in mcf10a cells results in an increased ratio of 28s18s by agilent bioanalyzer significance was calculatedby student™s t test in graphpad prism where �� p � f pax9 sirna depletion in mcf10a cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay a representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left protein was harvested after knockdown for hours using the indicated sirnas mock indicates no sirna and mock μmindicates no sirna and half the concentration of puromycin sint nontargeting was used a positive control quantitation of replicates using cellsof different passage numbers is shown to the right significance was calculated by oneway anova in graphpad prism where ���� p � and��� p � g pax9 depletion in mcf10a cells results in decreased 40s 60s and 80s ribosome subunit levels representative polysome profile ofmcf10a cells depleted using sirnas targeting either pax9 red or a nontargeting sint blue control equal amounts of protein were loaded oneach gradient this experiment was performed times using cells of different passage numbers101371 pgen1008967g001controlling gene expression [ ] in humans mutations in pax9 cause tooth agenesis aswell as hair loss [“] and reviewed in indeed pax9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia additionally mice homozygous for a partial deletion of pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth pax9 mice also exhibit a range of cardiac malformations another commonly impacted tissue in ribosomopathies while some research hasbeen done to identify the signaling pathways regulated by pax9 attempts to correct the developmental defects have been only partially successful [“] therefore further studies areneeded to identify all factors regulated by pax9 in order to understand pax9™s role in craniofacial developmentour work ties together pax9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by pax9 depletion we originally identified pax9 as a potential regulator of ribosome biogenesis in an sirna screen forproteins required to maintain nucleolar number probing pax9™s specific role in makingribosomes in human tissue culture cells we discovered that pax9 is required both for the prerrna processing that produces the small subunit 18s rrna and for global protein synthesiswe employed the genomewide transcriptomics analysis rnaseq to define pax9 dependentmrnas necessary for making ribosomes several of the differentially expressed mrnasincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerrna processing and global protein synthesis finally given an established rolefor pax9 in human craniofacial development we sought to model this disease in xenopus tropicalis x tropicalis embryos an established model of human ribosomopathies depletion ofpax9 in x tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structurex tropicalis embryos depleted of pax9 also show defective prerrna processing these resultsshed light on the plethora of factors whose expression is regulated by pax9 and open the doorto likely connections between pax9™s role in craniofacial development and human ribosomebiogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisresultspax9 depletion disrupts small subunit ribosome biogenesispreviously we performed an sirna screen for new regulators of nucleolar number in humanmcf10a cells we identified pax9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only fig 1b as in cells were fixed after hours of knockdown using pools of sirnas targeting sigfp as a negative control siutp4 as a positive control or sipax9 this transient knockdown approach successfully depleted pax9 s1a and s1b fig the mcf10a cells were then stained with anantibody to fibrillarin fbl a nucleolar protein to detect nucleoli and with hoechst todetect nuclei a cellprofiler pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in sigfp control cells to only in sipax9 and siutp4treatedcells we had previously used oligonucleotide deconvolution to additionally confirm thatpax9 depletion leads to reductions in nucleolar number using this approach individualdepletion of pax9 using of the sirnas from the original pax9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] as the sirna screen served as a phenotypic readout of nucleolar function we hypothesized that pax9 plays a role in humanribosome biogenesis through its function as an rnapii transcription factorwe sought to investigate the extent to which pax9 depletion affects human ribosome biogenesis using a panel of assays the first assay probes pax9™s role in rnapi transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] as pax9 is a known rnapii transcription factor it was relevant to test it for a possibleadditional role in rnapi transcription in this reporter assay the ratio of firefly luciferasewhich is under the control of the rdna promoter and measures rnapi transcription wasquantified relative to a renilla luciferase transfection control relative to a nontargeting control sirna sint sirnas targeting pax9 had no significant effect on rnapi transcriptionlevels after hours of knockdown in mcf10a cells fig 1c mock no sirna and sinol11were used as negative and positive controls respectively pax9 is therefore not required forrnapi transcription in mcf10a cellsnorthern blotting was used to define pax9™s role in prerrna processing in humans prerrna processing occurs via a number of different pathways fig 1d left and requires a number of transacting factors we therefore employed different probes to pinpoint any prerrna processing defects occurring after pax9 depletion in mcf10a cells s2a fig after hours of sirna knockdown pax9 depletion resulted in a significant increase in the 30s prerrna intermediate as well as a decrease in the levels of its 21s processing product relative tothe nontargeting sirna sint fig 1d middle and right and s2 fig additionally 41s levelswere decreased relative to the primary processing transcript 47s plus the 45s and 43s processing intermediates herein termed the primary transcript plus or ptp fig 1d middle andright and s2 fig quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by ratio analysis of multiple precursors ramp confirmed the statistical significance of these results fig 1d right and s2 fig these effects were also significant relative to a 7sl loading control fig 1d right and s2 fig because the 30s and 21sintermediates are both precursors to the 18s rrna pax9 is required for ssu biogenesis thesame prerrna processing defect was also detected in human embryonic kidney hek293ftand colon carcinoma rko cells depleted of pax9 indicating that pax9™s role in ribosomebiogenesis is conserved among diverse human cell lines s1 figbecause the prerrna processing defects indicate aberrant ssu biogenesis we sought todetermine the extent to which pax9 depletion affects the production of the mature 18s rrnaagilent bioanalyzer quantitation shows an increase in the ratio of 28s to 18s fig 1e genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesiscombined with the northern blot results indicating defects in the processing of the precursorsto the 18s rrna this result is consistent with a predicted reduction in 18s rrna levels takentogether these results argue that pax9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18s rrnawe also utilized a puromycin incorporation assay to test the extent to which pax9 depletion alters the final product of ribosome biogenesis global cellular translation fig 1f cellswith or without pax9 depletion are treated with a low dose μm of puromycin which isincorporated into all nascent peptides produced during a hour pulse western blottingfor incorporated puromycin shows decreased protein synthesis after hours of pax9 depletion relative to a nontargeting sirna control sint fig 1f mock μm puromycin withno sirna and mock at a halfdose of puromycin μm were used as negative controls fig1f these results confirm that pax9 depletion leads to reduced protein synthesis consistentwith a role for pax9 in ssu biogenesisbecause the above assays indicated a role for pax9 in small subunit prerrna processingand global protein synthesis we also tested the extent to which pax9 depletion is required forribosomal subunit biogenesis and assembly using polysome profiling we determined thatpax9 depletion does result in significantly decreased ssu 40s levels in mcf10a cells fig1g consistent with the reduction in 18s rrna levels seen in fig 1e additionally 60s and80s polysome fractions also showed significant decreases after pax9 knockdown fig 1ginterestingly both here and in previous experiments mcf10a cells do not demonstraterobust polysome fractions using this technique regardless we conclude that pax9 depletion results in decreased ssu biogenesis that impacts the assembly and function of theribosomeas disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which pax9 depletion changed the distribution of mcf10a cellswithin the cell cycle using flow cytometry s3 fig relative to a sint control depletion ofpax9 for hours resulted in a minor increase in the proportion of cells in g1 phase of thecell cycle but this was not statistically significant s3 fig sirnas targeting the ribosome biogenesis factor nol11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in g2 s3 fig consistent with previous findings inall these assays allowed us to conclude that pax9 regulates human ribosome biogenesisrnaseq analysis upon pax9 depletion reveals decreased levels ofnucleolar mrnas responsible for small subunit maturationas pax9 is a known rnapii transcription factor we hypothesized that pax9 works indirectlyto modulate ssu biogenesis fig 2a this is consistent with a nuclear but not nucleolar localization of pax9 in existing databases [“] pax9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins to test the hypothesis that pax9 affects nucleolar protein expression through itsfunction as a rnapii transcription factor we used rnaseq in mcf10a cells to define the setof mrnas that were differentially expressed after pax9 depletion relative to a nontargetingcontrol sirna sint pax9 depletion resulted in the differential expression of over mrnas fold change � or � q � fig 2b and s1 table approximately half of these were reduced in their levels consistent with the hypothesis that pax9 acts as a transcription factor to drive their expressionwhen considered as a whole the rnaseq dataset is enriched for several pathways knownto be regulated by pax9 s4a fig ingenuity pathway analysis of the differentiallyexpressed mrnas reveals enrichment of both wntca2 signaling differential expression of genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig rnaseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mrnas afterpax9 knockdown a schematic of how pax9 would act as a rnapii transcription factor to drive the levels of mrnas required for making the smallsubunit ssu of the ribosome in the cell nucleus pax9 binds to dna to affect the transcription of mrnas that either encode nucleolar proteinsdirect solid arrow or to transcribe mrnas that affect the levels of mrnas encoding nucleolar proteins indirect dotted arrows the resulting mrnasare translated in the cytoplasm into proteins that function in ssu prerrna processing in the nucleolus b rnaseq analysis after pax9 sirnadepletion in mcf10a cells reveals decreased levels of mrnas encoding nucleolar proteins relative to a nontargeting sirna control sint pax9depletion resulted in differential expression of mrnas fold change � or and fdr � of these mrnas had a decreased foldchange � and of those mrnas code for proteins designated as nucleolar in at least one of three databases [“] of the mrnas whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies c qrtpcr confirms reduced mrnalevels of the rnaseq candidates after pax9 sirna knockdown in mcf10a cells after depletion using sirnas targeting either pax9 or a nontargeting control sirna sint the levels of the indicated mrnas were quantified by qrtpcr using primers to each target gene relative to a 7slcontrol and sint data are shown as mean ± sem three replicates using cells of different passage numbers with technical replicates each wereperformed significance was calculated by oneway anova using graphpad prism where ���� p � d depletion of of the candidatemrnas rps6es6 rps9us4 rps28es28 and fbl individually results in the same prerrna processing defect as pax9 sirna depletion in mcf10acells representative northern blot after knockdown of the indicated sirnas using probe p3 a probe for the 7sl rna was used as a loading control prerrna processing intermediates detected by probe p3 are shown to the right of the northern blot ptp indicates the 47s 45s and 43s prerrnaprocessing intermediates e quantitation of northern blots using probe p3 as shown in fig 2d using ramp graph is mean ± sem n datawere analyzed using 2way anova in graphpad prism where ���� p � ��� p � and �� p � quantitation relative to the 7sl loadingcontrol is shown in s5 fig f sirna depletion of rnaseq candidates in mcf10a cells results in decreased global protein synthesis after hoursof knockdown with the indicated sirnas mcf10a cells were pulsed with puromycin for hour and protein was harvested western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left mock mock at half theconcentration of puromycin μm and sint nontargeting sirnas were used as negative controls g quantitation of three replicates usingmcf10a cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated sirnas relative to the sint andβ actin loading controls is shown as mean ± sem n significance was calculated by student™s ttest using graphpad prism where ���� p � ���p � and � p � 101371 pgen1008967g002 pathway members p x ˆ’ s4b fig and wntβcatenin signaling differentialexpression of pathway members p x ˆ’ s4c fig in cells depleted of pax9expression levels are increased for many of the mrnas in the wnt signaling pathway this isconsistent with previous results suggesting a role for pax9 in the negative regulation of wntsignaling [ ]as an additional validation of the rnaseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mrnas containmultiple potential pax9 binding sites making these mrnas candidates for direct transcriptional regulation by pax9 scanning for known pax9 binding sequences [™sgtcacgcwtgantgma3™ ™cgcgtgaccg3™ cd192ains ™gcgtgacca3™ and e5 ™gcggaacgg3™] in the kb upstream of the mrnas using centrimo analysis reveals and potential pax9 binding sites respectively in the kbupstream of the mrnas this number of potential binding sites upstream of the mrnas sites per gene is similar to that observed in pax9 chip experiments in the vertebral column of e125 mice sites per gene additionally centrimo enrichmentanalysis of the sequence kb upstream of each of the differentially expressedmrnas reveals significant enrichment of different dna binding sequences including thepax3 pax5 pax6 and pax7 dna binding domains as multiple pax proteins can bindthe same dna sequence this provides further evidence for pax9 regulation of these mrnas these analyses therefore support the hypothesis that pax9 regulates the levels of themrnas identified in our rnaseq datasetin the rnaseq dataset many of the differentially expressed mrnas have knownroles in nucleolar function for example have appeared in other genomewidesirna screens for nucleolar function s1 table [ ] additionally of the differentially expressed mrnas code for proteins designated as nucleolar in at least of nucleolar databases s1 table [“] this is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells surprisingly expression of most of the mrnas encoding nucleolar proteins genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis was downregulated upon pax9 depletion indicating that pax9 is requiredto maintain normal levels of many mrnas whose protein products are destined for functionin the nucleolus s1 tableto determine the mechanism of pax9™s function in ssu biogenesis we chose candidatesfrom the rnaseq dataset to follow up on in greater detail the mrna levels for the candidates were all downregulated after pax9 depletion and all code for nucleolar proteins fig 2band s1 table [“] four of the candidates rps6es6 rps9us4 rps28es28 and fblwere chosen on the basis of literature suggesting a role for these proteins in ssu prerrnaprocessing in hela cells [ ] additionally it was pertinent to analyze rpl5ul18 as it hasa known role in the p53dependent nucleolar stress response qrtpcr confirmed thernaseq results with reduced levels of each mrna when pax9 is depleted in mcf10a cellsfig 2c interestingly depletion of either rps9us4 or rps28es28 resulted in a decrease innucleolar number from “ to only in our original sirna screen therefore it is possible that the mechanism through which pax9 depletion results in decreased nucleolar numberrelies upon reduced expression of rps9us4 andor rps28es28 fig 2awe were able to confirm that depletion of of the tested candidates rps6es6 rps9us4 rps28es28 and fbl in mcf10a cells resulted in prerrna processing defects similarto that of pax9 depletion fig 2d and 2e s5 fig only rpl5ul18 did not give the 30sincrease characteristic of pax9 depletion although this was expected given its known role inlsu prerrna processing additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after pax9 depletion fig 2f and 2g puromycinincorporation was also reduced after rps6es6 depletion although it was not statistically significant fig 2f and 2g therefore pax9 may function as a transcription factor to directly orindirectly increase the expression of rps6es6 rps9us4 rps28es28 andor fbl fig 2aas the proteins encoded by these mrnas are required for ssu ribosome biogenesis fig their depletion after pax9 knockdown is a plausible mechanism through which pax9 regulates prerrna processing and global protein synthesisrnapii chipseq analysis reveals decreased transcription of mrnasencoding nucleolar proteins after pax9 depletionas the rnaseq analysis confirmed that levels of nucleolar mrnas were decreased afterpax9 depletion fig we sought to map how rnapii distributes on genes using rnapiichipseq as a readout of transcription through this approach we aimed to untangle the effectsof pax9 depletion on rnapii transcription from its effects on mrna stability since rnapiichipseq is able to detect genomewide changes in rnapii occupancy as pax proteins areable to both activate and repress target protein expression we have included genes withboth increased and decreased rnapii occupancy in this analysis approximately mrnaswere differentially occupied by rnapii upon pax9 knockdown compared to the nontargeting control sirna sint in mcf10a cells fold change cutoff � or � and maxtags � s2 table of these had decreased rnapii occupancy consistent with pax9 acting as a transcriptional driver of these mrnasto assess the validity of the rnapii chipseq dataset we again used centrimo to identifypotential pax9 dnabinding sites in the kb upstream of the genes with differentialrnapii occupancy searching for the ™ cgcgtgaccg ™ pax9 binding motif definedin revealed possible sites in the bp upstream of the differentially occupiedgenes also the known pax9 dna binding motifs cd192ains ™gcgtgacca3™ ande5 ™gcggaacgg3™ had and binding sites in these sequences respectively genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisadditionally analysis of motif enrichment ame identified the pax5 and pax6 dnabinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � since multiple pax proteins can bind the same motif this suggests thatthis dataset does contain mrnas that are regulated by pax9 of the differentially occupied genes have also been shown to be differentially regulated by pax9 directly in pax9chipseq experiments on e125 wt vertebral column murine tissue fig 3a and s2 table these analyses confirm the ability of rnapii chipseq to detect changes in rnapiimediated transcription after pax9 knockdownbased on the hypothesis that pax9 acts as an rnapii transcription factor for regulators ofnucleolar function fig we expected to detect changes in the rnapiimediated transcription of a number of mrnas encoding nucleolar proteins after pax9 depletion indeedmrnas coding for nucleolar proteins were enriched with of the genes with differentialrnapii occupancy coding for nucleolar proteins in at least one of three databases s2table [“] this is again higher than would be expected assuming that nucleolar proteins account for approximately of all cellular proteins additionally depletion of onegene with differential rnapii occupancy anln resulted in decreased nucleolar number inour sirna screen similar to the phenotype seen after pax9 depletion fig 1b notably of the genes with differential rnapii occupancy appeared in other genomewide screens for ribosome biogenesis factors s2 table [ ] another targeted screeninvestigated the effects of of the genes top2a and cdca8 on prerrna processingwhen depleted by sirna in hela cells however depletion of neither gave the 30s prerrna increase on northern blots characteristic of pax9 depletion in al

Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelial“mesenchymal transition EMT and apoptosis in BC are not fully understood We find that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4“ EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8“ Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspecific transmembrane protein foundin a variety of peripheral cells11“ is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored atˆ’ °C for western blotting and PCR analysis The othergroup of specimens was fixed in formalin and paraffinizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and verified for their authenticities by STR profiling Epirubicin was purchased fromPfizer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO “ “ and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The final protein staining score was the percentage score multiplied by the intensity score finalprotein staining scores were divided into three categoriesas follows negative “ low expression and “ high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturer™s instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments × cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were fixed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantifiedby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturer™s protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magnification × b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplan“Meier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magnification × f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell fluorescence was measured with a FACScan flowcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefficiency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of × stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacrificed by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups × stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacrificed by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were fixed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were fixed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunofluorescenceImmunofluorescence staining was performed as previously described19 In brief after corresponding treatments the cells fixed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfficial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by Kaplan“Meier analysis and thelogrank test was used to compare survival differencesbetween groups Pearson™s correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically significantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe first measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfficial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassignificantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and Kaplan“Meier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was significantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot significant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown significantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further confirm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efficiency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was confirmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1B“E Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown significantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunofluorescence showed the same results as thoseOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magnification × f Correlation analyses of proteinexpression levels between DNER and catenin g Kaplan“Meier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further confirm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were significantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunofluorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally Kaplan“Meier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfficial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a specific Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were significantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These findingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown significantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to confirm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efficiency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efficiency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantification of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see figure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightfieldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t

Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i

"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling œinsulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addison™s disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Ras“rapidly acceleratedtion offibrosarcoma Raf“mitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEK“extracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [“] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll “ percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx“ pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations “ percentiles for stimulating glucose uptake approx“ pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [“] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 œT polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctions”insulin œtoxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [“] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting œinsulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3K“AKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [“] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway Ras“MEK“ERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [“] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways œinsulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEK“ERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway œinsulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by “ []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by “ Ins2ˆ’ miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and “lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [“] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by ß cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces œinsulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEK“ERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [“] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[“] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ “] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[“] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ‰¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRS“PI3K“AKT anotheris the MAP kinases Ras“MEP“ERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant œinsulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no

"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adults™population of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [“] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [“] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients™ intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at ˆ’ °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients™ intakeUsual Macronutrients™ intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants™ physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrients™intake and physical activity between different FTO genotypes The post hoc Tukey™s test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrients™protein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [“] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ‰¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableˆ’Calorieˆ’ˆ’ˆ’ˆ’ATˆ’ˆ’ˆ’ˆ’P valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele ß ˆ’ kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrients™intakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [“] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients™ intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3“ and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors™ contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet “Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States “ JAMA “Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol “Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab 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institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes “ Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high 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HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul “ Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov “Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript 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among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [“] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patient™s health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patient™s quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett “ yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg “ aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol“ fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis “ holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal dis“quadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget “ nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology “20e1“ lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg “ easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol “ tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202“adenocarcinomastech coloproctol 0c'

since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal anti‚ammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [“] howeverthe findings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial fibrillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizen™shospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohn™s disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd findings and colonoscopicfindings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were defined as small and superficialmucosal disruptions denuded of villi figure ulcers weredefined as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients™ characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial fibrillationparoxysmal atrial fibrillationhypertensionhyperlipidemiacerebral infarctiondoac n years “dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n “mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis study™s findings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fisher™sexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients™ mean age was years range “ years and males and females participated in this study twenty patients had atrial fibrillation had paroxysmal atrial fibrillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range “ gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients refluxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy findingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansitefindingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [“] previous studies™ findings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [“] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe first quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce findings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal findings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the findings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudy™s findings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patients™ï¬ndings after the administration of the doacs and datadescribing the findings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patients™ï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its findings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conflicts of interest to disclose that arerelevant to this studyauthors™ contributionshiroyuki imaeda was responsible for the conception anddesign and final approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan œsmall bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda œsmall bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp “ j iwamoto y mizokami y saito œsmallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp “ h endo k hosono m inamori œincidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp “ a shiotani k haruma r nishi œrandomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp “ s j connolly m d ezekowitz s yusuf œdabigatranversus warfarin in patients with atrial fibrillation the newengland journal of medicine vol no pp “ m r patel k w mahaï¬ey j garg œrivaroxaban versuswarfarin in nonvalvular atrial fibrillation new england journal of medicine vol pp “ c b granger j h alexander j j v mcmurray œapixaban versus warfarin in patients with atrial fibrillation thenew england journal of medicine vol no pp “ c t ruï¬ r p giugliano e braunwald œcomparison ofthe efficacy and safety of new oral anticoagulants with warfarinin patients with atrial fibrillation a metaanalysis of randomised trials lancet vol no pp “ c s miller a dorreen m martel t huynh and a n barkun œrisk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp “1683e3 d caldeira m barra a ferreira œsystematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp “ t yamashita y koretsune y yang œedoxaban vs warfarin in east asian patients with atrial fibrillationan engageaftimi subanalysis circulation journal vol no pp “ m hori m matsumoto n tanahashi œrivaroxaban vswarfarin in japanese patients with atrial fibrillation circulation journal vol no pp “ y toya s nakamura k tomita œdabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp “ k kasai e ishida and y kobayashi œtwo cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp “ m okada and k okada œexfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23“e24 t vanassche j hirsh j ginsberg and j eikelboom œanspecific bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp “ h endo k hosono m inamori œcharacteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp “ i watari s oka s tanaka œcomparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal anti‚ammatory drugs a capsule endoscopystudy digestion vol no pp “ h endo k hosono t higurashi œquantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp “ d kaufman g leslie n marya œsmallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp “ a igawa s oka s tanaka œmajor predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c'

s case reports duplicate studies and those with insufficient available data were excludeddata extraction and managementdata were independently extracted by two investigators yao xl and wu ww according to the same inclusionand exclusion criteria disagreements were adjudicated by a third reviewer qu kthe following data will be extracted from eligible literatures the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509¢¢¢study characteristics name of the first author year of publication and sample size of included studiesparticipant characteristics tumor stage and age of patientsinterventions intervening methods and dosage administration route cycles and duration of treatment of huaiergranule¢ outcome and other data overall response rate orr disease control rate dcr overall survival osdiseasefree survival dfs quality of life qol immune indexes [cd3 cd4 cd8 natural killer cellsnk percentage and cd4cd8 cell ratios] and adverse effects we will attempt to contact the authors to request the missing or incomplete data if those relevant data are notacquired they will be excluded from the analysisquality assessmentto ensure the quality of the metaanalysis the quality of the included randomized and nonrandomized controlledtrials was evaluated according to the cochrane handbook tool and methodological index for nonrandomizedstudies minrrs supplementary table s2 respectively types of outcome measuresmain outcomesthe primary outcomes in present analysis included shortterm and longterm clinical efficacy and adverse effectsaccording to response evaluation criteria in solid tumors recist criteria i shortterm clinical efficacy the shortterm tumor response included orr and dcr orr was defined as thesum of complete and partial response rates and dcr was defined as the sum of complete response partialresponse and stable disease ratesii longterm clinical efficacy year os the time from the date of randomization to death from any cause year dfs the time from date of random assignment to date of recurrence or deathiii adverse events gastrointestinal adverse effects myelosuppression and hepatotoxicity secondary outcomesi qol qol was evaluated using the qualityoflife improved rate qir and karnofsky score kpsii immune function indicators the immune function of breast cancer patients was assessed in terms of cd3cd4 cd8 nk cells percentage and cd4cd8 cell ratiosstatistical analysisstata stata corp college station tx usa and review manager nordic cochran centre copenhagendenmark statistical software were used for statistical analyses cochrane™s q test and i2 statistics were used to assessheterogeneity among the studies if p01 or i2 a fixed effects model was used for the metaanalysisotherwise a random effects model was used the mantel“haenszel method will be applied for pooling of dichotomous data and results will be presented as risk ratio rr with their confidence intervals cis inverse variancemethod will be used for pooling of continuous data and results will be presented as standardized mean differencesmd with their cis a twotailed p005 was considered statistically significantthe presence of publication bias was investigated using the funnel plots begg™s and egger™s test if or more studiesare included in the metaanalysis [“] if publication bias existed a trimandfill method should be applied tocoordinate the estimates from unpublished studies and the adjusted results were compared with the original pooledrr sensitivity analysis was performed to explore an individual study™s influence on the pooled results by deleting onesingle study each time from pooled analysisresultssearch resultsthe initial search retrieved a total of s of which were excluded due to duplication after title and review s were further excluded because they were noncomparative clinical trials n19 were the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure study selection process for the metaanalysisnot related to huaier granule n9 were nonpeer reviewed s n8 were literature review or metaanalysisn3 and were case report and series n5 leaving studies as potentially eligible after detailed assessment offull texts studies with breast cancer patients n5 trials with insufficient data n8 and inappropriate criteriafor the experimental or control groups n11 were excluded ultimately trials [“] involving patients with breast cancer were included in the final analysis figure patient characteristicsall included studies were performed in different medical centers in china in total patients with breast cancerwere treated using conventional methods in combination with huaier granule while patients were treated usingconventional methods alone huaier granule was manufactured by qidong gaitianli pharmaceutical co ltd andgranted a manufacturing approval number issued by the chinese sfda z20000109 study and patient characteristics are summarized in table quality assessmentquality assessment of the risk of bias is shown in figure and table the results revealed that the literature retrievedfor the present study was of medium and high quality the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table clinical information from the eligible trials in the metaanalysisincluded studieschen qj chen y dai yg guo fd han sj lei ss liang yq li zh lu mq2017lu y qun sx ren xb shan cy tan zd tang y wang mh wang w wu yb xiong y xu f yang z2017yin x iiiiiiiviiiiinotprovidediiiiiiiiiviiiiiiviivnotprovidediiiiiiiiiiiiiiiiiiiiiiiiiviiiiiiiiiiiinotprovidedtumorstagepatientsconexpage year control vsexperimentalintervening methods“ range median ct vs cthuaier granules ˆ’ vs ˆ’ct vs cthuaier granulesoa meanoadosage ofhuaiergranulesduration oftreatments20gtime times monthcourse daycourses20gtime times weeks for a course daycourses months“ vs “ range ˆ’ vs ˆ’ ˆ’ vs ˆ’ meanmeanct vs cthuaier granules20gtime timesoadayct vs cthuaier granules20gtime times monthoadayct vs cthuaier granules20gtime times weeks for a course oadaycourses yearsnot providedct vs cthuaier granules20gtime timesoaday“ vs “ range ˆ’ vs ˆ’ ˆ’ vs ˆ’ meanmeanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times months for a course oadaycoursesct vs cthuaier granules20gtime times weeks for a course oadaycourses vs medianct vs cthuaier granules20gtime times weeks for a course not providedct vs cthuaier granules20gtime timesoadaycourses years mean ˆ’ vs ˆ’ ˆ’ vs ˆ’ ˆ’ vs ˆ’ meanmeanoadayct vs cthuaier granules20gtime times weeksoadayct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycoursesnot providednot providedct vs cthuaier granules20gtime times monthoadaynot providedct vs cthuaier granules20gtime times months ˆ’ vs ˆ’ meanoadayct vs cthuaier granules20gtime times monthsoaday“ range mean ct vs cthuaier granules20gtime times monthsoaday“ vs “ rangect vs cthuaier granules20gtime times weeks for a course “ vs “ range ˆ’ vs ˆ’ ˆ’ vs ˆ’ meanmeanoadayct vs cthuaier granules20gtime timesoadaycoursesnot providedct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycourses monthparametertypes1cid2 3cid21cid2 3cid25cid22cid22cid2 3cid25cid24cid2 5cid22cid2 3cid25cid22cid24cid2 5cid23cid2 4cid25cid24cid2 5cid21cid2 2cid24cid22cid2 3cid22cid2 3cid24cid25cid22cid23cid22cid22cid2 5cid25cid24cid2 5cid22cid22cid22cid21cid2 5cid22cid22cid2 3cid2zhang jg iiii“ vs “ rangect vs cthuaier granules20gtime times monthsoadayzhang y notprovidedzhao zw iiiiv“ vs “ range ˆ’ vs ˆ’ meanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times weeksoadayzhong sw zhou p iviiii“74range median ct vs cthuaier granules20gtime times monthsoaday“ vs “ rangect vs cthuaier granules20gtime times monthsoadaynotes control group conventional treatments alone group experimental group conventional treatments and huaier granule combined group1cid2 overall response rate and disease control rate 2cid2 overall survival or diseasefree survival 3cid2 adverse events 4cid2 quality of life 5cid2 immunefunction indexabbreviations ct conventional treatments oa oral administration the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure risk of bias summaryreview of authors™ judgments about each risk of bias item for included studies note each color represents a different level ofbias red for highrisk green for lowrisk and yellow for unclearrisk of biastable quality assessment of nonrandomized comparative studiesnonrandomized studiesadditional criteria in comparativestudytotalabcdefghijklstudyguo fdhan sjlei ssren xbwu ybyin xzhang yzhongsw zhou pa a clearly stated aim b inclusion of consecutive patients c prospective collection of data d endpoints appropriate to the aim of the study eunbiased assessment of the study endpoint f followup period appropriate to the aim of the study g loss to follow up less than h prospectivecalculation of the study size i an adequate control group j contemporary groups k baseline equivalence of groups l adequate statistical analysesnotes the items are scored not reported reported but inadequate and reported and adequatetherapeutic efficacy assessmentsorr and dcrfour clinical trials involving patients compared orr and dcr between the two groups as shown in figure the pooled results revealed that patients who underwent combination therapy experienced improved orr rr ci “ p002 and dcr rr ci “ p019 compared with those whoreceived conventional treatments alone although the dcr did not reach significant difference dcr p095 i2 was not heterogeneous among the studies therefore a fixedeffect model was used to analyze rr otherwise arandomeffect model was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of orr and dcr between experimental and control groupforest plot of the comparison of orr a and dcr b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined grouplongterm survival1year 2year 3year and 5year oseleven clinical trials with breast cancer patients reported os figure metaanalysis revealed that the 2yearrr ci “ p002 3year rr ci “ p00001 and 5year os rr ci “ p0004 of patients in the combined treatment group were significantly prolongedcompared with the control group there was statistical heterogeneity in 1year os p009 i2 and 2year osp00001 i2 according to the heterogeneity test therefore a randomeffect model was used to pool thismetaanalysis otherwise the fixedeffect model was used1year 2year 3year and 5year dfsten clinical trials with breast cancer patients reported dfs figure metaanalysis revealed that the 1yearrr ci “ p0003 2year rr ci “ p000001 3year rr ci p000001 and 5year dfs rr ci “ p003 of patients in thecombined treatment group were all significantly prolonged compared with the control group there was statisticalheterogeneity in 5year dfs p005 i2 according to the heterogeneity test therefore a random effectsmodel was used to pool this metaanalysis otherwise the fixedeffect model was usedqol assessmentfour trials with participants evaluated qir and three trials including patients reported kps data figure results demonstrated that the qol of breast cancer patients in the combined group was significantly better than thatof the control group indicated by significantly increased qir rr ci “ p000001 and kpsrr ci “ p000001 qir p084 i2 was not heterogeneous among the studiestherefore a fixedeffect model was used to analyze rr otherwise a randomeffect model was usedimmune function evaluationimmune status of the patients was examined between the two groups in eleven controlled studies including patients figure the percentages of cd3 cd3 rr ci “ p005 cd4 rr ci “ p000001 and nk cells rr ci “ p00001 and cd4cd8 ratio rr ci “ p000001 in the combined treatment group were significantly increased compared with the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of os between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year os d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of dfs between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year dfs d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of qol between experimental and control groupforest plot of the comparison of qir a and kps b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined groupthose in the conventional treatment alone group whereas the proportions of cd8 rr ˆ’ ci ˆ’to p033 did not differ significantly between the two groups a randomeffect model was used to pool thismetaanalysis due to significant heterogeneityassessment of adverse eventsas shown in figure patients treated with huaier granule and conventional methods exhibited lower incidences ofmyelosuppression rr ci “ p0001 and hepatotoxicity rr ci “p005 whereas analysis of gastrointestinal adverse effects rr ci “ p014 leukopeniarr ci “ p006 nausea and vomiting rr ci “ p052 andalopecia rr ci “ p020 did not differ significantly between the two groups there wasstatistical heterogeneity in gastrointestinal adverse effects p006 i2 according to the heterogeneity testand a random effects model was used to pool this metaanalysis otherwise the fixedeffect model was usedpublication biasas shown in figure the funnel plots begg™s and egger™s regression tests results showed that there was publicationbias in cd4cd8 ratio begg egger to determine whether bias affected the pooled risk ofcd4cd8 ratio a trimandfill analysis was performed the adjusted rr indicated a trend similar to the resultsof the primary analysis before p00001 after p00001 reflecting the reliability of the primary conclusionsparameters discussed less than papers were not conducted publication bias analysessensitivity analysisas figure signified the results revealed that no individual studies significantly affected the primary indicatorscd4 and cd4cd8 ratio which indicated statistically robust results parameters discussed less than paperswere not conducted sensitivity analysesdiscussionhuaier granule the active ingredient of huaier extract appears as a lightyellow powder through hotwater extractionethanol precipitation deproteinization and lyophilization procedures as a type of tcbp huaier granule hasbeen clinically applied as an effective adjuvant drug in cancer treatment for decades although several studies havereported that addition of huaier granule could be beneficial to patients with advanced breast cancer but the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of immune function between experimental and control groupforest plot of the comparison of immune function indicators including cd3 a cd4 b cd8 c and nk d cells percentage andcd4cd8 ratio e between the experimental and control group control group conventional treatment alone group experimentalgroup conventional treatment and huaier granule combined group the random effects metaanalysis model inverse variancemethod was used the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure comparisons of adverse effects between experimental and control groupforest plot of the comparison of adverse effects including gastrointestinal adverse effects a myelosuppression b hepatotoxicity c leukopenia d nausea and vomiting e and alopecia f between the experimental and control group control groupconventional treatment alone group experimental group conventional treatment and huaier granule combined group the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure funnel plot of cd4 a and cd4cd8 bexact therapeutic effects have yet to be systematically evaluated thus indepth knowledge of the efficacy and safetyof huaier granule is needed this systematic review will provide a helpful evidence for clinicians to formulate thebest postoperative adjuvant treatment strategy for patients with breast cancer and also provide scientific clues forresearchers in this fielddata from trials [“] including patients with breast cancer were included in our metaanalysishuaier granule in all of the included studies was manufactured by qidong gaitianli pharmaceutical co ltd thedosages of huaier granule were g per day via oral administration the pooled results revealed that the combinationof huaier granule and conventional treatment for breast cancer achieved more beneficial effects compared withthose treated solely with conventional therapy compared with conventional treatment alone huaier granule couldsignificantly improve orr and qol in patients with breast cancer p005 the study also assessed whether huaiergranule could prolong the longterm survival rates of breast cancer patients and the results showed that the and 5year os and and 5year dfs of patients were all significantly prolonged compared with the controlgroup these results indicated that using huaier granule could improve the short and longterm curative effects ofconventional treatment for breast cancert lymphocyte subsets cd3 cd4 cd8 cell subsets and cd4cd8 ratio and nk cells play an importantrole in antitumor immunity studies have shown that patients with advanced cancer showed decreased immunefunction and nk activity and exhibiting imbalance of t lymphocytes percentage many studies have reportedthat huaier granule can enhance the ability of the body™s immunity and resistance to tumors our analysisdemonstrated that the percentages of cd3 cd4 and nk cells and cd4cd8 ratio were all significantly increasedin breast cancer patients treated with huaier granule indicating that immune function of breast cancer patients wasimproved after huaier granule adjuvant therapysafety is the top priority of clinical treatment seven clinical trials with breast cancer patients reported adverse events according to world health anization standards metaanalysis revealed that patients who underwent huaier granule plus conventional treatment demonstrated a lower risk for myelosuppression and hepatotoxicitycompared with conventional treatment alone whereas analysis of other toxic side effects did not differ significantlytherefore huaier granule appears to be a safe auxiliary antitumor medicine for individuals with breast cancerthere were some limitations to our analysis currently five clinical trials table in which breast cancer are being treated by huaier granule in conjunction with conventional regimens have been registered onclinicaltrialsgov nct02615457 and nct02627248 and chinese clinical trial register chictr1800015390chictroic16007737 and chictrtrc11001250 however except for two studies most of the includedtrials were not registered before the first participant enrolled second as an important chinese patent medicinehuaier granule was mainly applied in china which may bring an unavoidable regional bias and subsequently influence the clinical application of huaier granule worldwide third different trials evaluated the treatment efficacy withdifferent outcomes resulting in a reduction in the size of the statistical sample making it difficult to summarize theresults at the same scale fourth several results demonstrated significant heterogeneity among the included trialswhich may be due to the different tumor stage tumor subtypes ages of the breast cancer patients and duration oftreatment however based on the currently available literature there are insufficient data to perform more statistical analysis to evaluate correlations in addition the efficacy of monotherapy of huaier granule in the treatment of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509figure sensitivity analysis for cd4 a and cd4cd8 b the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20202509101042bsr20202509table search results of clinical trial registrationregistrationnumbernct02615457nct02627248chictr1800015390chictroic16007737chictrtrc11001250titlephaseconditionsinterventionslocationshuaier granule intreating women withtriple negative breastcancerneoadjuvantchemotherapy with orwithout huaiergranule in treatingwomen with locallyadvanced breastcancer that can beremoved by surgeryhuaier granule forstage ii and iii triplenegative breastcancer with lymphnode metastasis amulticenterrandomizeddoubleblindplacebocontrolledclinical triala multicenter doubleblind randomizedplacebocontrolledstudy on stage iiiiitriplenegative breastcancer with lymphnode metastasistreated by huaiergranulesextract of fungi ofhuaier used for triplenegtive breastcancer”a prospectiverandomized controlledtrialivivtriple negative breasthuaier granuleqilu hospital ofcancershandong universityji™nan shandong chinabreast cancerhuaier granule otherqilu hospital ofchemotherapyshandong universityji™nan shandong chinaivtriple negative breasthuaier granulecancerthe first afliliatedhospital of amusouthwest hospitalchongqing chinaibreast cancerhuaier granuleivtriple negative breasthuaier granulecancersouthwest hospital thethird military medicaluniversity chongqingchinasouthwest hospital thethird military medicaluniversity chongqingchinabreast cancer also needs highquality evidence to verify however up to now huaier granule is mainly combinedwith radiotherapy chemotherapy or surgery and other conventional treatment methods for breast cancer we willkeep paying close attention to upcoming highquality clinical trials in our later studies and carry out further analyseson studies conducted huaier granule monotherapy against breast cancer finally publication bias was exists in someindicators which might because some authors tended to deliver positive results of s to editors therefore anyconclusions need to be made with cautionconclusionin summary findings of this metaanalysis indicate that the combination of huaier granule and conventional treatment is effective in treating patients with breast cancer the clinical application of huaier granule not only clearlyenhanced the therapeutic effects of conventional treatment but also effectively improved qol and immune functionin patients with breast cancer thus we anticipate that our study will provide valuable evidence for further evaluationof huaier granule on the other hand the low quality of some of the included publications increased the risk of biaswhich to some extent affects the reliability of this research therefore additional studies with highquality evidenceto verify the effectiveness of huaier granulemediated therapy for breast cancer are warrantedcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from national science foundation of china [grant numbers and ] the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20202509101042bsr20202509author contributionxi w and yao xl conceived and designed the methods yao xl and wu ww extracted the original data and drafted themanuscript yao xl wu ww and qu k performed statistical analysis xi w and yao xl interpreted results xi w and quk revised the manuscript all authors had full access to all data in the study and take responsibility for the integrity of the data andthe accuracy of data analysisabbreviationscbm chinese biological medicine database ci confidence interval cnki china national knowledge infrastructure csjdchinese scientific journal database dcr disease control rate dfs diseasefree survival kps karnofsky performance scoreminrrs methodological index for nonrandomized studies nk natural killer cells orr overall response rate os overallsurvival prisma preferred reporting items for systematic reviews and metaanalyses qir qualityoflife improved rate qolquality of life rct randomized controlled trial recist response evaluation criteria in solid tumors rr risk ratio sfdastate food and drug administration smd standardized mean difference tcbp traditional chinese biomedical preparationreferences ferlay j colombet m soerjomataram i mathers c parkin dm pi ˜neros m estimating the global cancer incidence and mortality in globocan sources and methods int j cancer “ 101002ijc31937 bray f ferlay j 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103322caac21397 wong ky tan ey chen jj teo c and chan pm the use of traditional chinese medicine among breast cancer patients implications forthe clinician ann acad med singapore “ zhu l li l li y wang j and wang q chinese herbal medicine as an adjunctive therapy for breast cancer a systematic review andmetaanalysis evid based complement alternat med wang w xu l and shen c effects of traditional chinese medicine in treatment of breast cancer patients after mastectomy ametaanalysis cell biochem biophys “ 101007s120130140348z mcpherson l cochrane s and zhu x current usage of traditional chinese medicine in the management of breast cancer a practitioner™sperspective integr cancer ther “ 1011771534735415607656 chen y wu h wang x wang c gan l zhu j huaier granule extract inhibit the proliferation and metastasis of lung cancer cellsthrough downregulation of mtdh jak2stat3 and mapk signaling pathways biomed pharmacother “101016jbiopha201802028 hu z yang a fan h wang y zhao y zha x huaier 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sarscov2 has resulted in numerous cases of coronavirus disease covid19 worldwide in addition to feverand respiratory symptoms digestive symptoms also are observed in some patients with covid19 angiotensinconverting enzyme ace2 was reported to be the receptor for sarscov2 the aim of this study was tocomprehensively investigate the digestive symptoms that occur in covid19 patients and the potential pathogenicroute of the sarscov2 infection in digestive tract ans from the oral cavity to the gastrointestinal tract weinvestigated the digestive symptoms of patients with covid19 and explored ace2 expression in digestive tractand lung cancers based on a series of bulk and singlecell rna sequencing data obtained from public databases wefound that of the patients with covid19 suffered from digestive symptoms among which pharyngalgia was the most common manifestation followed by diarrhea anorexia and nausea the bulktissue rna sequencing analysis indicated that digestive tract ans had higher ace2 expression levels compared tothe lung and the expression of ace2 in the lung increased with age singlecell rnaseq results showed that theace2positivecell ratio in digestive tract ans was significantly higher compared to the lung ace2 expression washigher in tumor cells compared to normal control nc tissues while in gastric tissues ace2 expression graduallyincreased from chronic gastritis to metaplasia to early cancer our data might provide a theoretical basis for screeningthe sarscov2 susceptible population and for the clinical classification of treatment of patients with covid19introductionthe global pandemic coronavirus disease covid19 caused by severe acute respiratory syninfection1 hasdrome coronavirus sarscov2been raging throughout the world by june covid19 has been found in countries with a totalof million confirmed cases worldwide includingover deaths2correspondence yujie liang yujie0350126com uiqing liao drliaoguiqinghotmailcom1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina2guangdong province key laboratory of stomatology no 2nd zhongshanroad guangzhou guangdong chinafull list of author information is available at the end of the these authors contributed equally jiabin xu mei chu fan zhongedited by i amelioit has been established that sarscov2 invadeshost cells by binding to the transmembrane receptorangiotensinconverting enzyme ace23 which also isthe receptor for sarscov and hcovnl6345 ace2plays a crucial role in the cellular entry for sarscov2which means that ace2positive cells may act as targetcells and are susceptible to infection6 thus the expression of ace2 might affect the invasion path and pathogenicity of the virusthe common symptoms of covid19 include feverfatigue cough myalgia and dyspnea7“ also somepatients may suffer from digestive system symptoms suchas pharyngalgia diarrhea nausea vomiting and abdominalpain10 suggesting that the digestive tract ans also maybe targeted by the virus studies have reported that ace2was expressed in lung esophagus epithelial cells ileum11colon12 kidney bladder13 and oral mucosa14 however the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cxu cell death discovery page of table demographics and clinical characteristics ofpatients with covid19total number of cases notable ace2 expression in aerodigestive cancerscancersample type samplesize nomeanexpressionlog2 fpkm standarddeviation “lungoralnctumornctumoresophagus ncstomachcolontumornctumornctumorage yearmedianrangeage group no years‰¥ yearssex nomalefemalecomorbidity nohypertensiondiabeteschronic obstructive pulmonary disease copdchronic renal diseasescardiovascular and cerebrovascular diseaseschronic liver diseasesmalignancydigestive system diseasesigns and symptoms nofevercoughdigestive symptomspharyngalgiadiarrheaanorexianauseafatiguedyspneainformation is lacking concerning the comparative analysisof ace2 expression in the entire digestive tract from theoral cavity through the gastrointestinal tract the oropharynx and gastrointestinaltract are physiologicallyinterlinked and share similar microbial environments15they also share similarities with respect to the microenvironment associated with epithelial carcinogenesisreports show that oral bacteria play a role in the genesis ofdigestive tract tumors16“ more than of covid19cases coexist with malignant tumors10 it is unclear whether there are differences in ace2 expression betweentumor and normal cellsin this study we assessed the clinical characteristicsand investigated the digestive symptoms of patientswith covid19 in guangzhou eighth people™s hospitalofficial of the cell death differentiation associationnote data was downloaded from the cancer genome atlas tcgaace2 angiotensinconverting enzyme nc normal control ace2 angiotensinconverting enzyme nc normal controlwe explored ace2 expression in digestive tract cancersand lung cancers based on a series of bulk tissue rnasequencing data from two independent databases andsinglecelltranscriptome data from three singlecelldatabases the results showed that nearly a quarter ofcovid19 patients had gastrointestinal symptoms thatace2 expression was higher in the digestive tract thanthe lung and the ratio of ace2positive cells in tumortissues was higher than that in paracancerous normaltissuematerials and methodspatients™ involvement and data collectionfortyeight hospitalized patients admitted from january to march in guangzhou eighth people™shospital the hospital exclusively designated for covid19patients in guangzhou who were clinically diagnosed withœviral pneumonia based on their clinical symptoms feveror respiratory symptoms with typical changes in chestradiology were preliminarily included in this studypatients without or with negative test results were excludedfrom this studydemographiccharacteristicsincluding medical history comorbidities signs andsymptoms were obtained from the electronic medicalrecord system of guangzhou eighth people™s hospital andanalyzed by three independent researchers all patientsincluded in our study provided consent for the nasopharyngeal swabs and clinicalinformation collectionthis study was approved by the institutional ethics boardof guangzhou eighth people™s hospital no and complied with the declaration of helsinki concerningmedical research using human subjectsinformationclinicalnucleic acid detection of sarscov2nasopharyngeal swabs were collected and placed intoa sterile tube containing rna preservation solution 0cxu cell death discovery page of fig the expression of ace2 in different tissues a“c ace2 expression of different tissues downloaded from tcga database a ace2 expressionin all samples b ace2 expression in tumor samples c ace2 expression in nc samples black solid line median value black dotted line interquartilerange d ace2 expression in cancer cell lines downloaded from ccle database solid line mean value dotted line median value ace2 angiotensinconverting enzyme tcga the cancer genome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinomahnsc head and neck squamous cell carcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinoma cclecancer cell line encyclopediathe swabs were sent for sarscov2 rna extractionand detection within h by a realtime reverse transcriptional polymerase chain reaction rtpcr systemby following the commercial test kit instructions da™angene cooperation cat da0930 as previously described19 briefly two pcr primer and probe sets targetingorf1ab and ncovn genes were separately added intothe same reaction tube positive and negative controlswere involved for detectionbulk tissue rna sequencing rnaseq data analysisthe rnaseq data level and clinical information forsamples were downloaded from the cancer genomeatlas tcgahttpsportalgdccancergovrepositoryusing the gdc data transfer tool the datasets used forace2 analysis included head and neck squamous cellcarcinoma hnsc esophageal carcinoma esca stomach adenocarcinoma stad colon adenocarcinomacoad lung adenocarcinoma luad and lung squamous cell carcinoma lusc gene expression waspresented as log2 fragments per kilobase per millionmapped reads fpkm the effects of age and sex onace2 expression were explored we also downloaded theboxplot for ace2 expression in human cancer cell linesfrom the cancer cell line encyclopedia ccle httpsportalsbroadinstituteccle database for verificationsinglecell rna sequencing scrnaseq data analysisthe lung cancer dataset exp0068 downloaded fromthe cancer singlecell state atlas httpbiocchrbmueducncancerseahomejsp and the lung tissue datasetsra878024 downloaded from the panglaodb httpspanglaodbseindexhtml were merged for further analysis the esophageal squamous cell carcinoma esccdataset gse81812 oral cancer dataset gse103322 gastriccancer dataset gse134520 and colorectal cancer datasetgse81861 were obtained from the gene expressionomnibushttpswwwncbinlmnihgovgeofor gse103322 cells from lymph nodes were removedthe r package seurat version was used for singlegeoofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by age a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by age top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomalogtransformed and scaledcell data analysis genebybarcode count matrices werenormalizedfollowed bydimension reduction using principal components analysispca uniform manifold approximation and projectionumap was used to carry out dimensionality reductionand clusteringall analyses were performed in r r version andgraphpad and the level of significance was set at p ‰¤ resultsdemographics and clinical characteristicsa total of patients diagnosed as covid19 wereincluded in the study twentyfive patients were male and were female the median patient age was years andranged from to years the majority of thepatients included in the study were under years of age of the patients had at least one underlyingcomorbidity the most common of which was hypertension three patients suffered from diabetes chronicobstructive pulmonary disease copd or chronic renaldiseasesrespectively two patients presented withcardiovascular and cerebrovascular disease one patienthad chronic liver disease malignancy or digestive systemdisease respectively the most common symptoms werefever or cough followed by digestive symptoms fatigue or dyspnea among patientswho presented with digestive system symptoms pharyngalgia was the most common manifestationfollowed by diarrhea anorexia and nausea table bulk tissue rnaseq data analysisa total of tumor samples and normal control nc samples were downloaded from the tcgadatabase table shows the expression of ace2log2fpkm in the different samples figure 1a“crespectively show ace2 expression in all tumor andnc samples from the different tissues the resultsshowed that digestive tract ans had higher ace2expression compared to the lung in digestive tractcancers ace2 expression gradually increased from theoral cavity to the esophagus stomach and the colonofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by sex a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by sex top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomafollowing the path of the digestive tract fig1a“c theresults were validated using the cancer cell line datafrom the ccle dataset fig 1dto analyze the effect of age or sex on ace2 expression thepatients were divided into a young group and an oldergroup ‰¥ based on age fig and the patients also wereseparated into females and males fig based on the clinicaldata in the tcga database the results revealed that for lungcancer based on either tumor samples or nc samples fig2a ace2 expression in the older group was higher comparedto the young group also for oral cancer the expression oface2 was significantly increased in the older group for thenc samples fig 2b as well as in the female group for thetumor samples fig 3bscrnaseq data analysissix different scrnaseq datasets were included in thisstudy sra878024 and exp0068 were lung normal tissueor lung cancer scrnaseq data respectively gse103322was oral cancer scrnaseq data gse81812 was scrnaseq data for the escc cell line kyse180 treated withdifferent doses of radiotherapy gse134520 includedscrnaseq data from chronic gastritis cg wild intestinal metaplasia wm severe intestinal metaplasia smand gastric cancer gc gse81861 included scrnaseqdata from colon cancer and adjacent normal tissues theresults showed that ace2positive cells accounted for thelowest proportion in lung samples with inthe nc group and in the tumor groupfig 4a the ace2positive rate in oral cancer cells was fig 4b while the ace2positive ratio inkyse180 treated with different doses of radiotherapywas for gy for gy and for gy fig 4c the ratios of ace2positive cells in the cg wm sm and gc groups were and respectively fig 4d the ace2positivecell ratio was in normal colonicmucosal cells and in colon cancer fig4e the ace2positive rate in the esophageal and oralmucosa was reported to be nearly and respectively we found that the ace2positivecell ratioofficial of the cell death differentiation association 0cxu cell death discovery page of fig see legend on next pageofficial of the cell death differentiation association 0cxu cell death discovery page of see figure on previous pagefig singlecell sequensing analysis of aerodigestive cancer cells a singlecell analysis of lung cancer cells tumor in exp0068 and nc cells insra878024 b singlecell analysis of oral cancer cells in gse103322 c singlecell analysis of escc cell line kyse180 tumor treated with different doses ofradiotherapy in gse81812 d singlecell analysis of cg wm sm and gc cells in gse134520 e singlecell analysis of colon cancer cells tumor and nc cells ingse81861 left column umap plots showing the distribution of cells colorcoded for pathology middle column umap plots showing the distribution oface2positive cell red right column stacked barplot showing the proportion of ace2positive cells red exp0068 was downloaded from the cancer singlecell state atlas sra878024 was downloaded from the panglaodb gse103322 gse81812 gse134520 and gse81861 were obtained from the geneexpression omnibus nc healthy normal control escc esophageal squamous cell carcinoma cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancer umap uniform manifold approximation and projection ace2 angiotensinconverting enzyme study were younger and presented with fewer underlyingdigestive system diseases the most common digestivesymptom was pharyngalgia which was consistent withother studies2224we observed that the expression of ace2 in the lungincreased with age but was independent of sex which wasconsistent with the report by chen 25 this maypartly explain why older patients with covid19 aremore likely to develop pneumoniathis study found that ace2 was highly expressed indigestive tract tumors or paracancerous tissues compared tothe lung for both the bulk tissue analysis and the singlecellanalysis elevated ace2 expression in the digestive tract suggested that the digestive tract ans also should be consideredto be vulnerable targets for sarscov2 infection it wasreported that sarscov2 might cause a cytokine storm andmultian failure in severe pneumonia patients26 similarlysarscov2 might interact with ace2 in digestive anscausing further damage to the mucous membrane barrier andincrease inflammatory cytokine production oralrelatedsymptoms are rarely reported with covid19 infectionsbut this does not indicate that an oral infection route forsarscov2 should be excluded the oral cavity and gastrointestinal ans share similarities in the microbiomeinflammation and tumorigenesis thus we expect that theoral and gastrointestinal ans should exhibit commoninteractions in the route of sarscov2 infection furthermore recent studies have reported a high positive rate ofsarscov2 detection in saliva2728indicating that oralinfection could be an early symptom of covid19interestingly we found that the ace2positive rate intumor cells was significantly higher compared to the ncgroup moreover ace2 expression in gastric tissuesgradually increased from chronic gastritis to intestinalmetaplasia to early gastric cancer similar findings werereported for the colon29 these results suggested thatcancer patients might have a higher risk of sarscov2infection from another perspective we suspected thatincreased expression levels of ace2 affected the occurrence of digestive tract tumors ace2 hydrolyzes ang iito ang1“ which negatively regulates the active reninangiotensin system ras reports show that ang1“plays an inhibitory role in the genesis and development offig ace2 expression increases along with the path of digestivetract ace2positive cell proportion in aerodigestive cancers ace2positive cell proportion in oral mucosa reported by xu 14 ace2positive cell proportion in esophageal epithelium reported by zou 11 ace2 angiotensinconverting enzyme nc healthy normalcontrol cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancerin digestive tract ans was significantly higher than inthe lung the ace2 expression also was higher in tumorcells compared to nc tissues the ace2 expression ingastric tissues gradually increased from chronic gastritisto metaplasia then cancer fig discussionthis study investigated the gastrointestinal symptoms of patients with covid19 who were admitted to theguangzhou eighth people™s hospital we explored ace2expression in digestive tract cancers and lung cancers basedon both bulk tissue rnaseq data and scrnaseq datathe median age of the patients was years whichwas similar to data reported by xu years21but was younger than the ages found in many otherreports892223 consistent with other recent reportshypertension and diabetes were the most commonunderlying comorbidities and fever and cough were themost common symptoms of covid19 infection923 inthis study of the covid19 patients exhibiteddigestive symptoms which was lower than the percentage reported by xu 21 and zhang 22 thereason for this difference may be that the patients in thisofficial of the cell death differentiation association 0cxu cell death discovery page of tumors3031 however sarscov2 could cause aninflammatory cytokine storm when the disease developsinto a longterm chronic state a potential risk mightdevelop for malignant changes to occur in the mucosaunder the effect of various cytokines therefore we suggest that when following patients with covid19 the riskof tumorigenesis should be consideredin summary the ace2positive cells in digestive tract tissuesmight provide possible routes for sarscov2 infectionace2 expression in lung tissue increased with age whichmight explain at least partially why older patients withcovid19 are more likely to develop pneumonia ace2expression was correlated to histological grading suggestingthat cancer patients might be more susceptible to sarscov future research should focus on whether the expression oface2 in digestive tract ans could affect the replication ofsarscov2 and whether sarscov2 infection could affectthe genesis or development of tumorsacknowledgementsthis work was supported by the guangdong financial fund for highcaliberhospital constructionauthor details1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina 2guangdong province key laboratory of stomatology no 2ndzhongshan road guangzhou guangdong china 3guanghua schoolof stomatology sun yatsen university 56th lingyuanxi road guangzhou guangdong china 4guangzhou eighth people™s hospitalguangzhou medical university guangzhou guangdong china5school of stomatology wuhan university 237th luoyu road wuhanhubei chinaconflict of interestthe authors declare that they have no conflict of interestpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreceived may revised july accepted july 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" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults™ activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged “ years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchison™sperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[“] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [“] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was ˆ’ °c lowest ˆ’ °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [“] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as œnonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb ‰ mets lpa“ mets and mvpa ‰¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package œcompositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemar™s test wasused to compare nonsnowfall and snowfall season™sproportions of those adhering to physical activity guidelines ie ‰¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of “ min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchison™s perturbation method [“] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained‰¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participant™s characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold “ yrsoldold ‰¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ‰¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [“] data where levels of adults™ physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [“ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors™ contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from ˜the policy research institute ministryof agriculture forestry and fisheries in japan™ and ˜the pfizer healthresearch foundation™ and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian government™s operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo “ japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet “kyu hh bachman vf alexander lt mumford je 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Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c'

" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ‰¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of “ afterresection [“] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies [“]the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [“] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ‰¤ had a cci of to and had a cci ‰¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ‰¥ plr ‰¥ and lmr ‰¤ respectivelyos was significantly shorter among patients with annlr ‰¥ than patients with an nlr in univariatehr [ ci “] p and multivariable hr [ ci “] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci “]p and multivariable hr [ ci “] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ‰¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ “] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ‰¥ independently predicted poor prognosis hr [ ci“] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn “overalln “age median range ynlr ‰¥ n “plr n pvalue “plr ‰¥ n “lmr ‰¤ n pvalue “lmr n “sex no femalemalerace no blackotherwhite bmi median range“““ ““ ““ “““ ““ “ “cci no ““‰¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ‰¥ demographicsn nlr n overalln nopvalueplr n plr ‰¥ n pvaluelmr ‰¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci “]p and diseasefree survival hr [ ci“] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci “] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ‰¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage‰¤ pathologic staget0t1t2t3cci“nlr ‰¥ plr ‰¥ lmr ‰¥ perineural invasionnoyesnananananananananana reference “ reference “ reference “nanananananananananananananacomplication grade “4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference “ reference “ reference “ “ “ reference “ reference “nananana reference “ “ “nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ “] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [“] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[“]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference “ reference “ “ “ reference “ reference “nananana reference “ “ “nanagenderfemalemalepathologic staget0t1t2t3cci“nlr ‰¥ plr ‰¥ lmr ‰¥ perineural invasionnoyesnananananananana reference “ reference “ reference “nanananananananacomplication grade “4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci “]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ‰¥ group anlmr was an independent predictor of poor prognosis hr [ ci “] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci “] p and plr hr [ ci“] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer center™s scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors™ contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin “ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j med“katz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol “neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet “oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama “chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer “proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer “bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity “ hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in 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cancer ann surg oncol“li w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther “ abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol “ quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell “ e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol “lausen b schaumacher m maximally selected rank statistics biometrics“lausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p “ mantovani a allavena p sica a balkwill f cancerrelated inflammationnature “ giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncol“li c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res “ nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol “ ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res “ zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta “ mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the

the diagnostic platform utilizing the detection of biomarkers in various body fluids called œliquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes efficient and reliable markerswithin the body fluids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ‚ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspecific to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue specific and reflect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the fieldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reflect the pattern specific for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentification of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artificialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas specific toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modifications of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and specificity in liquid biopsybasedmethods for disease detection and classificationthe patientthe5methylcytosine5mc modificationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespecific methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by “ translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor definitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeprofile of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodified at amino acid residues located on their n andcterminal tails these modifications act as epigenetic marksthat can specifically distinguish diseaserelated ccfdnas in bloodsamples various types of histone modifications are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspecific pcr methylationsensitivehighresolution melting and pyrosequencing garc­agimnez dna methylation specific to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modifiers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identified the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modifications on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodifications in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulfite sequencing rrbs meissner is an efficient method for absolute quantification ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulfite sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identified in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized profilingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodifications the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only “ of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantification of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body fluids are of clinical significance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ‚ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the blood“brain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 specificfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patient™s serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this field to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a œliquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations specific to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modifications patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identified as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic firmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspecific mirnas zhang mir was found to be a significant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was significantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was significantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentification ofrapidly developingsignificant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticefficacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnamplified neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be significantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularprofiling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound benefits recentlynovel immunological approaches produced insightful responsessnyder in addition the identification of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually benefit the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises “ ofthe maternal ccfdna wang andcan be efficiently detected atthe fifth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy down™s syndrome edward™ssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands efficient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modifications are specific to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the first reported use of fetalderivedepigenetic marker in maternal body fluids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspecific methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspecific dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the first such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been confirmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulfite treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna finds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microfluidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identification of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identified manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and

Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed women™s malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplan“Meier‘plotter database analysisKaplan“Meierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionP‘valueAge ‰ Tumor size ‰ Lymph node metastasis Present AbsentHistopathological grade I“II IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRT‘PCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ˆ’ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stably‘overexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine„¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures™ instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK‘ assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells × stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then × stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Student™s ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using Kaplan“Meierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by Kaplan“Meier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3c“f Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04c“q However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09c“f Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer c“q The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by Kaplan“Meierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010g“j and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ “] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro a“b The overexpression effect of GPX3overexpressed plasmid in breast cancer cells c“d Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells e“f Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network c“f The prognostic values of four miRNAs in breast cancer g“j The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pRˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ P‘valueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRP‘valuepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors™ contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics “ Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging “ Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol “ An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci “ Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res “ Kulis M Esteller M DNA methylation and cancer Adv Genet “ Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget “ Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res “ Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy “ Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol “ Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res “ Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie

natural killer nk cells are innate lymphocytes that play an important role in immune surveillanceagainst tumors and virusinfected cells and are an emerging target for tumor immunotherapynk cell immune surveillance is mediated by direct eï¬ector functions such as ifnÎ productionand cytotoxicity as well as immuneregulatory functions [such as interactions with dendriticcells “] of nk cells nk cells develop from precursors generated from hematopoietic stemcells hscs in the bone marrow and then begin the process of maturation before they egressto the periphery for immune surveillance since the initial discovery of nk cells in enormous progress has been made in our understanding of nk cell development and maturationas well as in identifying the cytokines and transcription factors that are important for theseprocesses nk cells that reach maturation represent the optimal functional status of the nk cellpopulation at steadystate and also represent the optimal functional status at the singlecell levelas evidenced by the upregulation of genes encoding cytotoxicityrelated eï¬ector molecules duringnk cell maturation compromised nkdependent immune surveillance usually accompaniesimpaired nk cell maturation therefore it is important to understand the molecularregulatory mechanisms underlying nk cell maturation which could potentially be exploited forthe development of novel therapeutic strategies in this review we aim to provide a framework ofthe current knowledge of nk cell maturation and the factors that regulate this process includingtranscription factors and cytokines and to discuss the importance of nk cell maturation inhealth and diseaseedited byzhigang tianuniversity of science and technologyof china chinareviewed byxi yanguniversity of manitoba canadajian zhangshandong university chinacorrespondencejiacheng bijcbisiataccnxuefu wangwangxuefuahmueducnspecialty sectionthis was submitted tonk and innate lymphoid cell biologya section of the frontiers in immunologyreceived june accepted july published august citationbi j and wang x molecularregulation of nk cell maturationfront immunol 103389fimmu202001945frontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationan overview of nk cellmaturationilc precursornk cell maturation in micein thein adult mice nk cells begin their developmentbone marrow and go through a stepwise cell diï¬erentiationprocess including hscs common lymphoid progenitors clpsprenk cell progenitors prenkps nk cell precursors nkpsimmature nk cells inks and mature nk cells mnksfigure clps are linˆ’il7rαckitsca1flt3 andcan generate prob and pret cells as well as the earliestcommon innate lymphoid cellcilcpprenkps representing an intermediate stage between clps andnkps are linˆ’cd244ckitlowil7rαflt3ˆ’cd122ˆ’ andcomprise both early nkcommitted precursors and il nkpsalso called refined nkps rnkps are linˆ’nk11ˆ’dx5ˆ’il7rαcd122nkg2d and diï¬erentiate into nk cells before the final nk lineage commitment nkps progress toan ink stage which is characterized by the acquisition ofnk11 and natural cytotoxicity receptor ncr expressionhowever nk cells at the ink stage are not yet functionallymature and their maturation continues in the bone marrowand the periphery the acquisition of dx5 and ly49 expressionmarks the maturation of nk cells to acquire optimizedcapacity nk cells continue a late maturation program whichis accompanied by an increase in their eï¬ector function andchanges in the expression of phenotype markers such asupregulation of cd11b cd43 and klrg1 or downregulation ofcd27 therefore based on the expression levels of cd27cd11bnk cell maturation can be divided into cd27cd11bˆ’cd27cd11b and cd27ˆ’cd11b stages nk cells maturefrom the cd27cd11bˆ’ink or m1 stage and then progressto the cd27cd11b transitional nk cell tnk or m2 stageand finally to the cd27ˆ’cd11b terminally mnk or m3stage during maturation nk cells gradually increase theircytotoxic capacity but decrease their potential for homeostaticexpansion nk cell maturation in humanshuman nk cells have developmental trajectories similar to thoseof mice figure hscs diï¬erentiate into clps and then nkps in humans the expression of cd122 on nkps is critical fornk cell lineage commitment the appearance of cd56 representsthe final step in the diï¬erentiation of nkps into nk cells thematuration of human nk cells can be divided into a cd56brightstage and a cd56dim stage based on the expression levels ofcd56 cd56bright nk cells are thought to be immature andcan diï¬erentiate into cd56dim nk cells with the acquisition ofcd16 while two subsets produce ‚ammatory cytokinescd56dim nk cells have more potent cytolytic activity cd56dimnk cells can further progress into latematuration stages withchanges in their surface markers and function the terminalmaturation of cd56dim nk cells with highest cytolytic activitycan be defined by the expression of cd57 approximately “ of all cd56dim nk cells in healthy adults express cd57on their surface interestingly highdimensional singlecell analysis can identify the high similarity between mousecd27ˆ’cd11b nk cells and human cd56dim nk cells andbetween mouse cd27cd11bˆ’ nk cells and human cd56brightnk cells additionally fu has showed that cd27 andcd11b can reflect distinct populations of human nk cells fromdiï¬erent tissues functionally similar with their counterparts inmice similarinnatelymphocytes the maturation of nk cells includes multiplephysiological processes to attain an optimal nk cell populationsize the maturation process usually requires the optimal egressof nk cells from the bone marrow and a finely tuned balancebetween survival proliferation and apoptosis at the steadystatemeanwhile optimal nk cell functional status at the singlecelllevel requires a dedicated transcriptional program dictated by anoptimal level of transcriptional factor activityto the diï¬erentiation process of othermodels used for investigation of nk cellmaturationbased on the above parameters several systems are availableto investigate the factors involved in the regulation of nk cellmaturationtool knockout mouse models provide a powerfultodetermine the eï¬ects of a geneofinterest on nk cellmaturation importantly an increasing number of studieshave employed nk cellspecific conditional knockout mousemodels in which cre recombinationdirected gene deletionoccurs soon after the acquisition of nkp46 “ thismodel allows gene deletion that is restricted to nk cells andgroup innate lymphoid cells ilc1s importantly italso allows the dissection of stagedependent eï¬ects elicitedby the geneofinterest on nk cell maturation adoptive transfer of nk cells into immunedeficient egil2rgˆ’ˆ’rag2ˆ’ˆ’ mice or reconstitution ofthe bonemarrow in lethally irradiated mice can recapitulate thematuration of nk cells under physiological conditions diï¬erent groups of nk cells can be monitored in acompetitive manner to assess cellintrinsic eï¬ects throughthe use of congenic markers such as cd4512 in vitro nk cell diï¬erentiation assays using op9 stromalcells provide an in vitro model to mimic cytokinedrivenphysiological nk cell diï¬erentiation from nk precursors this model also allows the determination of cellspecificeï¬ects associated with a geneofinterestseveral factors and pathways that play a role in nk cellmaturation have been identified using the abovementionedapproaches the results have demonstrated that nk cellmaturation is dependent on several critical signaling pathwaysand is triggered by a balance between extracellular signalscytokines and dictated by an optimal coordination oftranscription factor activity although nk cell maturation hasbeen extensively studied in mice knowledge about the factors thatcontrol human nk cell maturation remains limited neverthelessfrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationfigure dynamics of nk cell development and maturation in mice and human hematopoietic stem cells hscs differentiate into common lymphoid progenitorsclps and then differentiate into nk cell progenitors nkps the acquisition of cd122 marks nk cell lineage commitment from hscs inks generated from nkpscontinue to mature in the bone marrow and periphery for fully functional acquisition in mice cd27cd11b divides nk cell maturation into three stages in humancd56cd57 divides nk cell maturation into three stagesadvances in gene editing humanized mice models singlecell sequencing mass cytometry and genomewide associationstudies have led to a deeper understanding of how nk cellmaturation is regulated in humanscytokines that regulate nk cellmaturationincreasing evidence suggests that multiple cytokines are involvedin nk cell development table for instance il7 scf andflt3l are critical for cd122 nkp generation from hscswhile il15 is essential for nk cell lineage commitment andmaturation from cd122 nkps to mnk cells additionallymultiple cytokines have been found to be involved in nk cellmaturation by modulating il15 signalingil7 scf and flt3lstromafree culture has shown that preculture in il7 scf andflt3l is necessary for nk cell development by inducing il responsiveness in progenitors clps normally exhibithigh levels of il7 receptor scf receptor and flt3l receptorhowever the expression of these receptors is gradually lost duringthe process of nk cell maturation nevertheless these cytokineshave distinct roles in nk cell development for instance nk cellmaturation is almost normal in il7rα or il7deficient mice whereas the homeostasis of thymusderived cd127nk cells is exclusively dependent on il7 showing characteristicssimilar to those of human cd56highcd16ˆ’ nk cells il7promotes the survival of cd56bright nk cells by increasing bcl2expression although it does not increase nk cell cytotoxicityinterferongamma ifnÎ production or the expression ofactivation markers il7 alone is not sufficient to supporthuman nk cell development as evidenced by the findings inhuman il7 knockin nod scid gamma nsg mice scfpromotes the survival of peripheral ckit nk cells and theabsence of ckit signaling reduces the generation of nk cells fromfetal liver precursors additionally a marked deficiency ofnk cells has been observed in the spleen of mice lacking flt3l flt3l not only potently induces il15 responsivenessin progenitors but can also significantly expand nk cells byincreasing the number of il15expressing cd11chi dendriticcells dcs indicating that flt3l is important for the earlydiï¬erentiation of nk cells and nk cell expansionlymphotoxinlymphotoxins alpha ltα and beta ltβ belong to thetumor necrosis factor tnf ligand superfamily ltα boundto the ltβ receptor ltβr constitutes a membraneboundltα1β2 heterotrimer that is essential for secondary lymphoidfrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationtable factors involved in nk cell maturationfactorseffect stages affectednotereferences clp nkpˆ’ nkp ink mnk nkp ink mnk nkp ink mnk nkp ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk± inkrequired for il15 responsivenessessential for development but limits maturationinduce expression of tbet gata3 blimp1 and id2downstream of il15 receptorsuppress ebox genes suppress socs3promote s1pr5 ifng repress eomesrepress tbetpromotes tbet expressioninduced by tbetinduced by tbet zeb2 ko phenocopy tbet kodispensable for early nk cell developmenttgfβ independentpromote autophagy or suppress tbet ˆ’transcription factorse4bp4tcf1ets1stat5id2tbeteomestox12prdm1zeb2gata3smad4foxo1cytokinesil7 scf flt3llymphotoxinil15il17tgfβthis table lists transcriptional factors or cytokines that either promote œ or suppress œˆ’ nk cell maturation the stages affected clp nkp ink or mnk as well asthe functions or features of these moleculescritical for nkp generation from hscessential for earlystage nk developmentpromote nk development maturation activation survival and homeostasisinduce socs3cell cycle arrest before mature stage nkp nkp nkp ink mnkˆ’ ink mnkˆ’ nkp ink mnk“ “““““ ““ tissue anogenesis as evidenced by defective lymph nodedevelopment and altered splenic microarchitecture in ltaˆ’ˆ’ltbˆ’ˆ’ and ltbrˆ’ˆ’ mice ltα and ltβ are expressed inactivated lymphocytes including t b and nk cells whereasltβr is expressed exclusively in nonlymphoid tissues includingin bone marrow stromal cells the ablation of ltα or ltβrleads to impaired earlystage development of nk cells “ bone marrow stromal cells from ltaˆ’ˆ’ or ltbrˆ’ˆ’ micecannot efficiently support early nk cell progenitors il15can overcome the arrest of ltaˆ’ˆ’nk cell development in vitro however nk cell progenitors from wildtype mice generatedmore nk11 cells than nk cell progenitors from ltaˆ’ˆ’ micein the presence of il15 these findings suggest that theinteraction between ltα on nk precursors and ltβr on stromalcells creates a permissive microenvironment that is essential forthe earlystage development of nk cellsil15il15 belongs to the Îc family of cytokines sharing a Îcchain with il2 and il15 signals through il15rβÎc encoded by il2rg heterodimers either alone withintermediate affinity or as a complex with il15rα with highaffinity a phenotype with a nearcomplete loss of nkcells is observed in the absence of either il15rα il15rβ Îcchain or il15 “ in contrast mice deficient for il2 or have normal nk cell development and maturationduring nk cell development il15 acts from cd122nkpsuntil the terminal maturation of nk cells indeed the ablationof il15 does not aï¬ect the production of prenkps or nkpsalthough il15 is widely expressed in various tissues and bymany cell types irf1dependent il15 production in the bonemarrow microenvironment is critical for nk cell generation importantly the eï¬ects of il15 on nk cell development andmaturation require transpresentation of il15 by il15α onbone marrowderived dcs “ in an il15αdosedependentmanner in addition il15 transgenic mice or mice thatoverexpress il15 have a dramatic increase in the number ofmnk cells il15 is also needed to support mnk cell survivaland promote mnk cell expansion in the steadystate similarwith the observations in mice il15 determines human nk cellmaturation and homeostasis which is supported by evidencefrom patients with Îc mutation and humanized mice as well asby experiments involving nk cell ex vivo developmentil17il17a is a pro‚ammatory cytokine that also plays animmunosuppressive role in some settings such as in tumorsby recruiting myeloidderived suppressor cells promotingangiogenesis or suppressing cd8 t cells in addition ourgroup recently found that il17 signaling negatively regulatesnk cell maturation and function ifnÎ production and thecytolytic activity of nk cells as well as nk cell antitumor andantiviral immune activity are enhanced in il17aˆ’ˆ’mice although total nk cell numbers are comparable with thosein wildtype mice increased percentages of terminal maturecd27ˆ’cd11b nk cells were observed in il17aˆ’ˆ’ il17f ˆ’ˆ’il17aˆ’ˆ’il17f ˆ’ˆ’ and il17raˆ’ˆ’ mice overexpression of il17a in vivo reduces cd27ˆ’cd11b nk cell subsets mechanisticallyil17a signaling suppresses il15inducedphosphorylation of stat5 via the upregulation of socs3 in nkcells leading to inhibition of nk cell terminal maturation on the other hand il17a exerts contextdependent eï¬ects onnk cells il17 signaling is required for ifnÎ production andthe cytolytic activity of nk cells derived from lipopolysaccharidefrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationlpsprimed mice as well as for optimal production ofgranulocytemacrophage colonystimulating factor gmcsfby nk cells in fungal infection which plays a nonredundant rolein activating neutrophils for fungal control tgfβisgrowth factor betatgfβtransforminga majorimmunosuppressive cytokine many cell types express tgfβand nearly alllymphocyte populations express the tgfβreceptor tgfβr deletion of the tgfβ receptor subunit tgfβrii enhances mtor activity and the cytotoxic activity of nkcells in response to il15 in the steadystate nkspecificdeletion of tgfβrii in ncr1cre tgfbr2flfl mice has a minimaleï¬ect on conventional nk cell maturation and homeostasis in contrast constitutive tgfβ signaling arrests nk cellmaturation moreover tgfβ blocks il15induced mtoractivation thereby suppressing the activation and functionsof nk cells consistent with the observations in micetreatment with tgfβ in vitro inhibits cytokinestimulatedmetabolism of human nk cells via canonical tgfβ signaling in contrasttransgenic mice expressing a dominantnegative form of tgfβrii under the control of the cd11cpromoter cd11cdntgf β rii exhibit an increase in the numberof mature nk cells in the periphery in an in vitro assayfor the diï¬erentiation of nk cell progenitors to less maturecd122nk11 and more mature nk11dx5 nk cells in thepresence of il15 and op9 stromal cells the addition of tgfβmarkedly blocked the derivation of mature nk cells from wildtype precursors whereas precursors from the cd11cdntgf β riimice were not aï¬ected mechanistically tgfβ was foundto mediate nk cell immaturity during ontogeny by arresting thecell cycle of nk cells at the least mature cd11bˆ’cd43ˆ’ andintermediate cd11bcd43ˆ’ stages as well as by limiting nkcell transition at the terminally mature cd11bcd43 stage as a result of resistance to tgfβ infant cd11cdntgf β riimice are protected against murine cytomegalovirus mcmvinfection in addition smad3 has been found to suppresse4bp4mediated nk cell development and eï¬ector functions asevidenced by the increased number of mnk cells in smad3ˆ’ˆ’mice and the increased levels of granzyme b il2 and ifnÎ insmad3ˆ’ˆ’nk cells meanwhile the silencing of smad3 inthe human nk92 cell line allows for the upregulation of e4bp4which subsequently promotes ifnÎ production thediï¬erences in phenotypes between cd11c cells and nkp46cells following blockade of tgfβr signaling might be due to theabrogation of the tgfβr signal at diï¬erent stages of nk celldevelopmentmaturation further studies are required to revealthe temporal regulation of nk cell maturation by tgfβtranscription factors thatregulate nk cell maturationnatural killer cell development is also regulated by sequential andcoordinated transcription factor activity figure for instancetranscription factors such as e4bp4 tox and id2 are requiredfor nk cell lineage commitment while id2 tbet eomes andfigure transcriptional regulation of nk cell development and maturationmultiple transcriptional factors mediate regulation at distinct stages during nkcell development and maturation e4bp4 tcf1 and id2 are essential for nkcell lineage commitment tbet eomes tox prdm1 gata3 zeb2 andsmad4 are critical for nk cell maturation the discrepancy of foxo1 in nkcell maturation needs further confirmationzeb2 are required for nk cell maturation transcription factorsthat mediate nk cell maturation usually promote the expressionof genes coding for eï¬ector molecules receptors responsible foregress and cellsurface maturation markerse4bp4e4bp4 is expressed in clps e4bp4ˆ’ˆ’ mice specificallylack nk cells indeed the numbers of prenkps rnkpsink cells and mnk cells but not those of clps are reducedin the bone marrow of e4bp4ˆ’ˆ’ mice ectopic expression ofe4bp4 in e4bp4ˆ’ˆ’clps can rescue nk cell production these findings indicate that e4bp4 acts at the clp stage and isrequired for nk cell lineage commitment prenkps and rnkpsare not aï¬ected in the absence of il15 signaling however theabsence of e4bp4 results in the failure of il15responsive nkpproduction specifically il15 cannot rescue nk cell productionin e4bp4ˆ’ˆ’ bone marrow in contrast ectopic expressionof e4bp4 enables limited nk cell production even in the absenceof il15 signaling however ectopic expression of eomesid2 or tbet cannot rescue nk cell production in the absenceof e4bp4 furthermore e4bp4 was found to directly regulatethe expression of eomes and id2 while the histone h2adeubiquitinase mysm1 has been found to be critical for therecruitment of e4bp4 to the id2 locus and be required fornk cell maturation but not nk lineage commitment inaddition notch1 has been identified as an e4bp4 target genein nk cells the abrogation of notch signaling blockednk cell production similar to that observed in e4bp4ˆ’ˆ’miceexposure to notch peptide ligands at an early stage rescuedthe defective nk cell development from e4bp4ˆ’ˆ’ progenitors moreover sumoylation and phosphorylation can regulatee4bp4 activity and then ‚uence nk cell development the ablation of either sumoylation or phosphorylation sitessignificantly increased the transcriptional activity of e4bp4 andpromoted the production of nk cells therefore not only thefrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationexpression level but also the activity of e4bp4 is critical fornk cell development however the regulation of e4bp4 activityby posttranslational modifications needs further investigationfirth reported that specific ablation of e4bp4 in eitherink or mnk cells had no eï¬ect on nk cell lineage maintenanceand homeostasis therefore these findings confirm thate4bp4 is required for nk lineage commitment but not for thematuration from the ink to the mnk stage or the survival ormaintenance of mnk cells although it has been reported that theupregulation of e4bp4 can enhance ifnÎ production in nk92cells the roles of e4bp4 in human nk lineage commitmentand development are not well definedtcf1tcf1 encoded by tcf7 gene is a member ofthe highmobility group hmg of proteins and is important for t celldevelopment and function tcf1 was initially found to bind tothe ly49a promoter and be required for the acquisition of ly49aexpression during nk cell development loss of tcf1 resultsin a greater than reduction in the levels of ly49d on nkcells subsequently studies on a tcf1 reporter mouse straindemonstrated that tcf1 was expressed by prenkps nkps inkcells and mnk cells but not clps in the bone marrow prenkps nkps and mnk cell numbers were reduced in tcf7ˆ’ˆ’mice moreover detailed analysis in splenic nk cells showed thattcf1 expression was high in cd27cd11bˆ’ nk cells declinedwith nk cell maturation and disappeared in cd27ˆ’cd11b nkcells surprisingly the number of terminally mature nk cells intcf7ˆ’ˆ’ mice is significantly increased these findings indicatethat tcf1 is essential for nk cell development but limits nkcell terminal maturation in humans tcf1 has been found tobe uniquely expressed in circulating cd56bright nk cells andnot in cd56dim nk cells the eï¬ects of tcf1 on humannk cell development and maturation as well as the underlyingmechanisms need further investigationets1ets1 is the founding member of the ets family of wingedhelixturnhelix transcription factors and is highly evolutionarilyconserved ets1 has been shown to function from the prenkpstage and is required for the expression of various transcriptionfactors including tbet and id2 activating nkrs includingnkp46 ly49d and ly49h and signaling molecules thelevels of mnk cells are significantly decreased in the bonemarrow and spleen of ets1ˆ’ˆ’mice moreover ets1ˆ’ˆ’mnk cells display impaired eï¬ector function and decreasedexpression of activating nkrs but increased expression ofinhibitory nkrs meanwhilein human nk cellstaveirne revealed that ets1 induced the expression oftranscription factors such as tbet gata3 and blimp1 thatdetermine nk cell development suggesting that ets1 mightplay a similar role as in humans moreover il2 and il can increase ets1 expression through erk12 signaling inhuman nk cells these observations indicate that ets1is essentialthe stagespecific requirement of ets1 for nk cell maturation requiresfurther investigationfor nk cell development howeverstat5downstream of il15 receptor jak1 associates with il2rβleading to stat3 phosphorylation while jak3 associates withil2rÎc resulting in stat5 phosphorylation the jak“stat5 pathway has been identified as being essential fornk cell maturation germline deletion of jak1 or stat5 andconditional deletion of jak1 or stat5 in nkp46 cells both lead toa marked reduction in nk cell levels as well as inhibition of nkcell maturation in the bone marrow and the periphery consistent with the reduced levels of mnk cells jak1floxfloxncr1icre tg or stat5floxfloxncr1icre tg mice showed defective nkcelldependent tumor surveillance moreover stat5 tetramersare required for the maturation of nk cells from the ink tothe mnk state in bone marrow and spleen as evidenced by thesubstantial decrease in mnk cell numbers but normal levelsof nkps and ink cells in stat5astat5b tetramerdeficientdouble knockin mice stat5b mutation also leads todefective human nk cell maturation and impaired lytic function these observations indicate that jak13stat5 is criticalfor nk cell maturationid2id2 is a member of the inhibitor of dnabinding id proteinfamily which acts by preventing eproteins such as e2a e2 and heb from binding ebox canntgcontaining targetgenes through heterodimerization id2 is expressed at high levelsafter nk cell lineage commitment and in all subsequent stages id2 was first reported to play an essential role in thegeneration of peripheral lymphoid ans and nk cells throughthe use of id2ˆ’ˆ’ mice although id2 deficiency does notreduce linˆ’cd122 nk cell progenitors in the bone marrow itdoes lead to reduced nk cell numbers and impaired maturationof nk cells as evidenced by the decreased expression of cd43and cd11b maturation markers as well as granzyme expression a more recent study showed that the hematopoietic deletionand acute deletion of id2 or id2 deletion in nkp46 cellsresults in the loss of most nk cells this indicates that id2 isrequired for nk cell maintenance at all stages of developmentid2 suppresses ebox genes required for nk cell maturationsuppresses socs3 expression and is required for normal il15receptor signaling strong il15 receptor stimulation by il2 preligated to an antiil2 antibody or socs3 deficiency partiallyrestores homeostasis in id2deficient nk cellsin support of the role of id2 in nk cell maturation deficiencyof mysm1 which mediates the recruitment of e4bp4 to the id2locus leads to defects in nk cell maturation but not nk lineagespecification or commitment mysm1 also plays a role inmaintaining an open chromatin structure at the id2 locustbet and eomestbet tbox expressed in t cells encoded by tbx21 is amember of the tbox family of transcription factors that areprimarily expressed by t cells and nk cells in tbetˆ’ˆ’ mice thenumber of nk cells is significantly decreased in the spleen liverand peripheral blood but modestly increased in bone marrownk cells these defects result from the impaired egress offrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationnk cells from the bone marrow tbet directly regulates s1pr5by binding to its locus and loss of tbet leads to thedecreased expression of s1pr5 in addition tbetdeficientnk cells express lower levels of the maturation markers cd43cd11b and dx5 and higher levels of ckit and integrin alphavwhich reflects the immaturity of nk cells therefore tbet isessential for nk cell maturationanother tbox transcription factor eomes eomesoderminalso plays nonredundant roles in nk cell maturation in micemost spleen nk cells express eomes expression of eomes marksnk cell maturation and eomes nk cells are characterized bythe expression of dx5 and the absence of trail expression eomes is required for nk cell maturation hematopoieticdeletion of eomes in eomesfloxfloxvavcre mice results in asubstantial reduction in the number of nk cells in the spleenand blood and to a lesser extent in the liver lymph nodes andbone marrow loss of eomes also renders nk cells phenotypicallysimilar to eomesˆ’ nk cells derived from wildtype mice withexpression of trail but lacking dx5 as well as decreased levelsof ly49 receptors and cd11b in addition trail inks requireeomes for conversion to dx5 mature nk cells following theirtransfer into il2rgˆ’ˆ’rag2ˆ’ˆ’ mice eomes is also required tomaintain nk cell maturity as temporal deletion of eomes inmature nk cells results in the loss of the maturation marker dx5and upregulation of traildespite the critical roles of eomes and tbet in nk cellmaturation loss of either transcription factor does not markedlyimpair the eï¬ector functions of nk cells at the singlecell levelin vitro further confirming that these factors have roles in nkcell maturation but not in nk cell identitytoxthe tox thymocyte selectionassociated hmg box proteinbelongs to a family of evolutionarily conserved dnabindingproteins besides tox family members include tox2 tox3and tox4 the tox protein is highly expressed in ink andmnk cells in the bone marrow the loss of tox significantlyreduces the frequency and number of mnk cells in the peripheryand bone marrow with a severe block in the transition from theink to mnk stage and to a lesser extent from the nkp to theink stage id2 expression is significantly reduced in the nk cellsthat remain in toxˆ’ˆ’mice however id2 expression does notrescue the defective maturation of toxˆ’ˆ’nk cells in additionyun demonstrated that tox enhances the maturationof human nk cells and aï¬ects though not directly regulatestbet expression during nk cell maturation this indicatesthat tox is required for nk cell maturation both in humansand in mice however the regulatory circuit downstream oftox during nk cell maturation requires further investigationsbesides tox tox2 has been found to play a critical rolein human nk cell maturation vong found that tox2is preferentially expressed in human nk cells among severalimmune cell populations and is upregulated during human nkcell maturation tox2 is essential for human nk cell maturationand cytotoxicity tox2 directly upregulates tbet expressionand tbet overexpression can rescue the tox2 knockdownmediated nk cell maturation defects these results implythat tox2 functions upstream of id2 in human nk cellshowever the role of tox2 in murine nk cell maturationremains to be elucidated combined these results show tox andtox2 play a crucial role in nk cell maturationprdm1the expression of the transcriptional repressor b lymphocyteinduced maturation protein blimp1 also known as prdm1can be induced by il15 stimulation and its expressionis tbetdependent blimp1 is constitutively expressed bynk cells and is upregulated during nk cell maturation in bothhumans and mice loss of blimp1 aï¬ects nk cell homeostasisleading to increased nk cell numbers in the bone marrow andlymph nodes and reduced nk cell numbers in the liver and thelungs loss of blimp1 also leads to impaired nk cell maturationas shown by the decreased levels of the cd27ˆ’cd11b nkcell subset and expression of the maturation markers cd43and klrg1 however blimp1 is dispensable for most eï¬ectorfunctions of murine nk cells and even suppresses the productionof ifnÎ tnf and ltαin human nk cells by directly bindingto multiple conserved regulatory regions an impairedresponse to il15 in the absence of blimp1 might be the causeof the aberrant maturation of blimp1ˆ’ˆ’ nk cellszeb2tb so synergizes with zeb2 to promote nk cell maturationtbet is necessary and sufficient to induce zeb2 expression andzeb2 expression positively correlates with nk cell maturation zeb2 is required for normal nk cell homeostasis deletionof zeb2 in nk cells results in the retention of nk cells in thebone marrow and reduced nk cell numbers in the peripheryzeb2 overexpression leads to a reduced number of nk cellsin the bone marrow moreover mice with nkspecific zeb2deletion lack mature cd27ˆ’ nk cells while zeb2 overexpressionincreases the numbers of mature cd27ˆ’ nk cells the reducednumbers of mature nk cells might be due to poor survivaland response to il15 as well as the downregulation of s1pr5expression in zeb2deficient nk cells additionally the levels ofthe maturation markers klrg1 and cd146 are also proportionalto the levels of zeb2 expression this implies that zeb2 promotesnk cell maturation interestingly although the absence of zeb2in nk cells compromises the overall nk cellmediated immuneresponse as evidenced by the increased susceptibility of nkzeb2ˆ’ï¿

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'

"decreased expression of Hsp70 caused by ibuprofen amplified the activation of caspase-9 significantly compared with that of Bax. Furthermore the similar increase in the activation of cisplatin-dependent Bax and release of cytochrome c by ibuprofen suggests that Hsp70 also inhibits the post mitochondrial steps between the release of cytochrome c and the activation of caspase-9. If Hsp70 were acting downstream of the mitochondria one would predict that it interferes with the activation of caspase-9 in response to cytochrome c either by inhibiting the formation of the apoptosome or by preventing the binding of pro-caspase-9 to this complex. When we studied the effects of Hsp70 on the formation of and recruitment of pro-caspase-9 to the apoptosome the cell lysates were immunoprecipitated although Hsp70 failed to migrate with Apaf-1 cytochrome c or caspase-9 (data not shown). These results may be supported by previous report that no association between Hsp70 and Apaf-1 or apoptosome complex was observed even under in vitro activation of caspase by the addition of cytochrome c and dATP.44 Furthermore we were unable to identify a new target for Hsp70 in the process of caspase-9 activation indicating that its inhibitory activity is attributable to another indirect effect instead of a direct one as previously reported. Altogether the data presented here are the first evidence of cell death inhibition by Hsp70 by its targeting of both upstream and downstream mitochondrial processes while the precise mechanisms by which it interferes with the activation of caspase-9 remains to be clarified. In ibuprofen potentiated the antitumoural properties of cisplatin in the cells of lung adenocarcinoma via a mechanism of action mediated by the suppression of Hsp70. These findings may promote the development of a new strategy to increase the effectiveness of cisplatin in the treatment of NSCLCs as well as highlight the putative merits of developing anticancer treatments targeting Hsp70. Materials and Methods Materials The mouse monoclonal anti-Hsp70 the rat monoclonal anti-Hsc70 and rabbit polyclonal HSF-1 antibodies purchased from Stressgen “ Enzo Life Sciences Inc. Plymouth Meeting PA USA. Anti-Bax rabbit polyclonal (N-20) and anti-VDAC-1 goat polyclonal (N-18) antibodies were purchased from Santa Cruz Biotechnology Inc. Santa Cruz CA USA. Anti-cytochrome c mouse monoclonal antibody (556433) was obtained from BD Pharmingen Inc. San Diego CA USA. Anti-caspase 9 and -ERK antibodies were acquired from Cell Signaling Technology Inc. Danvers MA USA. The mouse monoclonal antibody against actin was obtained from Chemicon International Inc. Temecula CA USA. Anti-Bax 6A7 monoclonal antibody and other reagents were purchased from Sigma-Aldrich St. Louis MO USA. Cell culture and viability assay A549 and H358 lung cancer cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum at 37?°C. BEAS-2B cells were grown in bronchial epithelial basal medium. All NSAID and cisplatin were dissolved in dimethyl sulphoxide and added to the medium at indicated concentrations. The activity of mitochondrial dehydrogenase 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay was used to measure cell death/survival. The reaction product was measured at A570 and the relative viability of cells treated with reagents versus untreated cells was calculated. TUNEL staining The TUNEL assay was performed using an in situ cell death detection kit (F Hoffmann-La Roche Basel Switzerland) according to the manufacturer's instructions. The ratio of TUNEL-positive cells to the total number of cells was calculated. Immunoprecipitation and cell fractionation A549 cells were lysed in RIPA buffer (50?mM Tris-HCl pH 7.5 150?mM NaCl 1?mM sodium orthovanadate 1?mM EDTA 0.1% NP-40 10?mM NaF) containing the Calbiochem Protease Inhibitor Cocktail Set III (Merck KGaA Darmstadt Germany). The cell lysates and immunoprecipitates were resolved in Laemmli sample buffer. The samples underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis were transferred to a polyvinylidene difluoride membrane reacted with the respective antibodies and detected with an ECL chemiluminescence detection kit (GE Healthcare Fairfield CT USA). For the immunoprecipitation the cell lysates were incubated with the indicated antibodies for 1?h at 4?°C. Protein G-sepharose beads were added to collect the immunocomplexes for an additional 1?h of incubation. The pellets were washed three times with lysis buffer. The mitochondria and cytosol fractions were prepared as described previously.45 Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed as described previously46 using an EZ ChIP kit (Upstate Biotechnology Inc. Waltham MA USA). Briefly after adding formaldehyde A549 cells were suspended in SDS lysis buffer and the chromatin DNA was disrupted by sonication. For the immunoprecipitation the lysate was incubated with anti-HSF-1 antibody followed by immobilization on salmon sperm DNA/Protein G agarose. The protein/DNA complexes extracted with elution buffer were heated to 65?°C for 6?h to reverse cross-links then digested with proteinase K. DNA fragments were amplified in PCR with the ChIP assay primers containing the heat shock element sites in human Hsp70 promoter. PCR primers for the ChIP assay were as follows: Hsp70 (?103/+7) (F) 5?-TGATTGGTCCAAGGAAGGCT-3? and (R) 5?-AAAAAGGTAGTGGACTGTCGC-3?. Reverse transcriptase-PCR RT-PCR was carried out using a Qiagen (Valencia CA USA) One-Step RT-PCR Kit. We used the following primer pairs to amplify. Hsp70 (F) 5?-ATGAAGCACTGGCCTTTCCA-3? (R) 5?-TTGTTCTGGCTGATGTCCTT-3? Hsc70 (F) 5?-TGGAACTATTGCTGGTCTCAA3? (R) 5?-AGAACCACCAACCAGGACAAT-3? HSF-1 (F) 5?-TTCGACCAGGGCCAGTTT-3? (R) 5?-AGAGCTGGCCACAGCATCA-3? actin (F) 5?-AGAGGCATCCTCACCCTGA-3? (R) 5?-CATCTCTTGCTCGAAGTCCA-3?. The products were examined by agarose gel electrophoresis after 23 cycles. RNA interference The sequences of the sense strands used to generate specific siRNA were obtained as follows: HSF-1 5?-AAGTACTTCAAGCACAACAA-3? 5?-AAGAGTGAAGACATAAAGAT-3? 5?-AAGTCGTCAACAAGCTCATT-3?. The siRNAs were synthesized using the Silencer siRNA construction kit (Ambion; Applied Biosystems Inc. Carlsbad CA USA). Double-stranded Hsp70 and control siRNA duplex were synthesized as followed by Qiagen: Hsp70-specific sequence 5?-CCAUUGAGGAGGUAGAUUAdTdT-3?. A549 cells were transfected with each siRNA (10?nmol/l) using the Lipofectamine 2000 (Invitrogen; Applied Biosystems Inc.) and grown for 72?h to allow an effective decrease in the expression of the respective target molecules. Quantification of apoptosis by flow cytometry A549 cells were washed with Annexin V staining buffer (10?mM HEPES pH 7.4 150?mM NaCl 5?mM KCl 1?mM MgCl2 1.8?mM CaCl2) and incubated with CF488A-Annexin V and propidium iodide (Biotium Inc. Hayward CA USA) in staining buffer for 30?min at 37?°C in the dark. Fluorescence was measured using a FACSCalibur (BD Biosciences San Jose CA USA) and the data were analyzed with CellQuest software (BD Biosciences). JC-1 staining and quantification A549 cells were cultured at 37?°C for 48?h on glass chamber slides and treated with ibuprofen and cisplatin at the specified concentrations for 48?h. Mitochondrial permeability transition was determined by staining the cells with 55?66?-tetrachloro-1133?-tetraethyl- benzimidazolylcarbocyanin iodide (JC-1; Molecular Probes Invitrogen Carlsbad CA USA) in the dark. The cells were subsequently washed with assay buffer according to the manufacturer's protocol and immediately imaged using a fluorescence microscope (Keyence Corporation Osaka Japan) with the red (?excitation: 560±40?nm band pass filter ?detection: 630±60?nm band pass filter) and green (?excitation: 470±40?nm band pass filter ?detection: 535±50?nm band pass filter) fluorescence channels. Flow cytometric analysis was assayed with the JC-1 Mitochondrial Membrane Potential Kit (AAT Bioquest Sunnyvale CA USA) according to the manufacturer's directions using a FACSCalibur and the results were analyzed by CellQuest software. In vitro casapse-9 activity determination Caspase-9 activity was measured by a fluorometric assay in whole-cell lysates using Ac-Leu-Glu-His-Asp-MCA substrate (Peptide International Inc. Louisville KY USA). A549 cell extracts were mixed with Ac-LEHD-MCA in ICE standard buffer (100?mM HEPES pH 7.5 10% sucrose 0.1% CHAPS 10?mM DTT 1?mM PMSF) and cleavage of the fluorogenic peptide substrate was monitored at 37?°C for 30?min by a SPECTRA max GEMINI EM (Molecular Device Sunnyvale CA USA) fluorometer with excitation at 370?nm and emission at 460?nm. This work was supported in part by a Grant-in-Aid (23591477) from the Ministry of Education Culture Sports Science and Technology of Japan. Apaf-1 apoptotic protease-activating factor 1 COX cyclooxygenase HSF-1 heat shock factor 1 MTT 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide NSAID nonsteroidal anti-inflammatory drug NSCLC non-small cell lung cancer PCR polymerase chain reaction RNAi RNA interference RT reverse transcriptase TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling Edited by G Raschellà The authors declare no conflict of interest. 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Lung cancer has high mortality often accompanied with systemic metabolicdisorders The present study aimed at defining values of transnodules crossclinical phenomic and lipidomic network layers in patients with adenocarcinoma ADC squamous cell carcinomas or small cell lung cancer SCLCWe measured plasma lipidomic profiles of lung cancer patients and found thataltered lipid panels and concentrations varied among lung cancer subtypes genders ages stages metastatic status nutritional status and clinical phenomeseverity It was shown that phosphatidylethanolamine elements and were SCLC specific whereas lysophosphatidylcholine and snposition1 and phosphatidylcholine and were ADCspecific There were statistically more lipids declined in male ages latestage metastasis or body mass index Clinical transomics analyses demonstrated that one phenome in lung cancer subtypes might be generated from multiple metabolic pathways and metabolites whereas a metabolic pathway andmetabolite could contribute to different phenomes among subtypes althoughthose needed to be furthermore confirmed by bigger studies including larger population of patients in multicenters Thus our data suggested that transomic profiles between clinical phenomes and lipidomes might have the value to uncoverthe heterogeneity of lipid metabolism among lung cancer subtypes and to screenout phenomebased lipid panels as subtypespecific biomarkersK E Y WO R D Slipidomics lung cancer phenomes subtypes transomicsINTRODUCTIONLung cancer is a systemic and aggressive disease withhigh morbidity and mortality and it is often accompanied with systemic metabolic disorders for exampleup or downregulated expression of mechanismassociatedgenes or activation of metabolismdependent enzymes Forexample metabolismassociated genes of small cell lungThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e151101002ctm2151wileyonlinelibrarycomjournalctm2 of 0c of ZHU et alcancer SCLC cells altered after mitogenactivated proteinkinase MAPK kinase module MEK5ERK5 was blockedaccompanied by dysfunctions of several lipid metabolismpathways like the mevalonate pathway for cholesterolsynthesis1 Lipids mainly including subclasses of phosphatidic acid PA phosphatidylcholines PC phosphatidylethanolamine PE phosphatidylglycerol PGphosphatidylinositol PI and phosphatidylserine PShave multiple important biological functions such asbiomembrane composition vesicular trafficking adhesion migration apoptosis energy storage neurotransmission signal transduction and posttranslational modification They have alterations under circumstance of lungcancer Circulating levels of PCs and PEs in patients withnonsmall cell lung cancer NSCLC differed from thosewith noncancer lung diseases or health and were suggested as diagnostic biomarkers of early NSCLC2 Theheterogeneity of circulating lipidomic profiles was foundto exist among patients with squamous cell carcinomasSCC adenocarcinoma ADC or SCLC and there was aclear correlation between genomic and lipidomic profilesof lipidassociated proteins and enzymes3 As the part ofclinical transomics the lung cancerspecific and subtypespecific lipidomics in the circulation were defined and evidenced by integrating lipidomic data with genomic expression of lipid proteins among lung cancer subtypesClinical transomics is defined as a new subject tointegrate clinical phenomes with molecular multiomicsfor understanding molecular mechanisms of diseases inmultiple dimensions4 Clinical transomics becomes moreimportant as a new and novel approach for the discovery of diseasespecific biomarkers and therapeutic targetsalthough there are still many obstacles to be overcomefor example specificity and decisive role of transnodulesamong multiomic networks for intra and intercellular communication56 Recent studies applied the transomics among phosphorproteomics transcriptomics genesequencing and genomics for new molecular category ofliver cancer to provide a new therapeutic strategy7 As thepart of clinical transomics clinical lipidomics was considered as one of major metabolic profiles for identificationand validation of lung cancerspecific biomarkers by integrating clinical phenomes with lipidomic profiles89 Clinical lipidomics could demonstrate the complexity of thelipidome in metabolic diseases and lung cancer and presented the variation among diseases and subtypes of lungcancer1012Our previous study demonstrated the difference oflipidomic profiles among patients with different lungcancer subtypes and the potential association betweenlipidomic phenotypes and gene expression oflipidmetabolismassociated proteins and enzymes as a conceptevaluation3 The present study furthermore investigatedthe values of transnodules crossclinical phenomic andlipidomic network layers in the recognition of lung cancersubtypes ADC SCC and SCLC in order to understandclinical phenomeassociated lipid changes or lipidomicphenotypeassociated clinical phenomes We also evaluated the differences of lipidomic profiles between maleand female various ages early and late stages with orwithout metastasis body mass index BMI or and digital evaluation scores less or more than METHODS AND MATERIALSChemical agentsThe internal standard cocktails were subscribed fromAvanti Lipids Polar Alabaster AL USA the acetone acetonitrile ammonium bicarbonate dithiothreitol formicacid iodoacetamide and Tris base Analytical Gradefrom SigmaAldrich St Louis MO USA and ammonium acetate NH4OAc hexane isopropyl alcohol IPAmethanol and highperformance liquid chromatographygrade chloroform CHCl3 from Merck Millipore Billerica MA USAPatient populationThe study designed as a casecontrol approach wasapproved by the Ethical Evaluation Committee of Zhongshan Hospital ethical code B2018187 The subjectsgave informed consent for clinical data collection andlipids analysis before all the other procedures The studyincluded lung cancer patients diagnosed according topathology of whom were ADC SCC SCLC and other healthy people The stage and severity of lung cancerwere defined according to the Eighth Edition of TNMClassification for Lung Cancer13 Patients were recruitedduring October to March Healthy controlsparticipated were blood donors in Zhongshan HospitalSubjects with other respiratory diseases or family historyof lung cancer were excluded Fasting blood was drawnfrom healthy controls and lung cancer patients on theday of entering hospital to harvest plasma All the clinicaldata including symptoms signs laboratory tests imagespathologic information and survival status years laterwere collected and followed upDigital evaluation score systemThe Digital Evaluation Score System DESS is a scoreindex system by which clinical descriptive information of 0cZHU of each phenome can be translated into clinical informatics14When the severity of each component was scored as or of which represented the most severe condition whereas indicated normal physiological range Thegross DESS scores ranged from to points the higherthe score the severer the condition A total of clinical phenomes were collected and scored in each of threelung cancer group including histories symptoms signs laboratory measurements image features and pathologic indexes as listed in Table S1spectrometry analysisLipid extraction for massAbout µL plasma was collected into a glass tubeinto which µL internal standard was added and then mL of methanolchloroformformic acid asreported previously315 This mixture was incubated at “—¦C overnight after vigorous shaking Two milliliters ofHajra™s reagent M H3PO4 M KCl were droppedblended and centrifuged at rpm for min Afterstratification chloroform in the lower layer was pipettedto another glass tube and concentrated to µL withthe nitrogen flow where the liquid with isopropyl alcoholhexane100 mM ammonium acetate at the ratio of was added till mL The sample was then centrifugated at rpm at —¦C for min The normalphaseliquid chromatography coupled TripleQuadrupole massspectrometer QTRAP SCIEX Framingham MAUSA was used for lipid extraction by the positive and negative electrospray ionization mode In the multiple reactionQTrap was utilized to scan the precursorproduct ion andexamine the mode operation Each test was repeated threetimes The peak area of each pair was quantified with multiple reaction monitoring data by the software MultiQuantAB SCIEXPurification of plasma lipidsLipid samples were derived through Ultimate SiO2 mm × mm µm Agilent Technologies Santa ClaraCA USA with mLmin flow rate highpurity heliumIn the meanwhile µL was added with the split ratio of at the ignition chamber temperature of —¦C and theinjection port temperature of —¦C It was started at temperature —¦C which gradually increased —¦Cmin to—¦C and kept for min The mass spectrometry was subjected to liquid chromatographymass spectrometer analysis FOCUS DSQTM II Thermo Fisher Scientific mainlyunder the following conditions Electron Ionization EI asionization source ion source temperature at —¦C ionization voltage at eV multiplier voltage at kV minsolvent delaying and amu of scan rangeIdentification of lipidomic profilesLipid extracts were loaded onto an Ultremex silica column mm × mm µm which was fitted with a mm× mm silica guard cartridge Phenomenex TorranceCA USA and then eluted The sample was enriched ata gradient of nLmin In the min™s run B phasewas from to min then rose to from to min linearly ramped for min as to return from to min until the end The QTrap was conductedin the multiplereaction monitoring mode and the different precursorproduct ion pairs were scanned in thepositivenegative electrospray ionization mode Up to lipids of plasma samples were carried out to get possiblelipids chemical structureslipidomic profilesComprehensive analyses ofMultiQuant software AB SCIEX was used to process dataafter lipids were identified by mass spectrometry Further MetaboAnalyst software wwwmetaboanalystcawas utilized for conducting multivariate statistical analysis cluster analysis dimensionality reduction and makingheat mapphenome and lipidome network layersTransnodule analyses crossThe typespecific lipids were identified as more than twotimes elevated or declined significantly compared withother lung cancer subtypes fold change and Pvalue whereas the coexpression lipids were identified as those similarly changed in all lung cancer subtypesas compared with healthy controls The expression quantitative trait locus eQTL model was utilized to evaluatetransnodules between lipidomic profiles and clinical phenomesStatistical analysisData were presented as mean ± SE The means of eachgroup were used for calculation and comparison Statistical significance of differences between two groupsor among multiple groups was determined by Student™sttest or oneway ANOVA test respectively Statistical 0c of ZHU et alsignificance was affirmed when Pvalue We alsoseparately calculated mean values of each phenome™sDESS score in different lung cancer subtypes which wereranked to obtain top clinical phenomes of those threegroups of patients Volcano maps showed the significantlyelevated or declined lipids in ADC SCC or SCLC patientsA VIP plot was further exploited to sort the lipids according to their importance to differentiate the four groups Toexplore the correlation between lipid elements and clinical phenomes we applied the lipidquantitative trait locimodel modified from eQTL model Besides MatrixlQTL Rpackage was used to acquire the significant phenomelipidpairs and corresponding Pvalues Moreover GraphPadPrism was utilized to make the receiver operating characteristic curve to evaluate the earlydiagnostic value andaccuracy of clinical phenomespecific lipid elements inADC SCC or SCLC The present study furthermore analyzed the significant differences of lipids among differentages eg and between female and maleearly and late stage metastasis and nonmetastasis highand low DESS scores ‰¤ and and high and lowBMI ‰¤ and RESULTSwith lung cancer subtypesClinical phenomes of patientsEighteen female and male lung cancer patients wereenrolled in the present study aged from to ± years old including ADC SCC and SCLC The totalscores of DESS were ± ± and ± in patientswith ADC SCC and SCLC respectively The DESS values of SCLC group were significantly higher than those ofhealthy control group P Top clinical phenomesof ADC SCC or SCLC patients as well as patients survivedor nonsurvived during study period were listed in Table Stages at primary diagnosis and recruitment period for thestudy lymphatic metastasis N12 in ipsilateral paratracheal hilum or mediastinum and enhanced images egfocus enhanced in CT or hypermetabolism in PETCTwere shown in all three subtypes of lung cancers In addition thyroid transcription factor1 TTF1 Napsin A keratin and location of tumor were noticed in ADC obscureboundary emphysema tumor size the cycle number offirst line chemotherapy obstructive pneumonia atelectasis and pulmonary nodule in SCC as well as number ofmetastatic lymph nodes in SCLC separately Top clinicalphenomes were similar between survived and nonsurvivedpatients but the total amounts of DESS of nonsurvivedpatients were significantly higher than those of survivedpatients Table Of total clinical phenomes hadthe statistical significance of each two groups inbetweenTable P or lesswith lung cancer subtypesLipidomic profiles of patientsTotal lipid elements of plasma were identified qualitatively and quantitatively mainly including PAs PCs PEs PGs PIs PSs lysophosphatidylcholineslysoPC lysophosphatidylethanolamines lysoPE lysophosphatidylglycerols lysoPG lysophosphatidylinositols lysoPI lysophosphatidylserines lysoPS ninediacylglycerides and triacylglycerols TAG Levels ofsome lipid elements in ADC SCC or SCLC patients weresignificantly higher Table S2 or lower Table S3 as compared with healthy control twofold P The majority of those elevated lipid elements were PC PA and lysoPC in ADC PE PC PS and PG in SCC or PS PE PG lysoPS and lysoPI in SCLC Of those declinedlipid elements were PS in ADC whereas and were PA in SCC and SCLC respectively Table demonstrates lung cancer subtypespecific lipid elements identified by those lipid elements elevated or declined exclusively in each lung cancer subtype for example some oflysoPC and PC in ADC whereas lysoPI lysoPS PE andPA in SCLC By partial least squares discrimination analysis PLSDA analysis top lipid elements were definedon the basis of variable import in project VIP score of eachgroup TAG565 lysoPG182 and PG and increased in ADC lysoPG181 PA140245 PI384180PA and and PE385PE180 increased inSCLC and PI362PI180 and lysoPG182 decreased in SCCdetail in Figure 1A There was a clear distribution oftop lipid elements among lung cancer groups as compared with the healthy control Figure 1B Of those significantly increased lipid elements in patients with lungcancers top six lipids of each group were identified Figure levels of LysoPC sn2 sn1 and sn1 in ADC Figure 2A PS363 in SCC Figure 2B andPA and and PI and inSCLC Figure 2C were significantly higher than in otherthree groups PLSDA component analysis demonstratedthat five principal components selected were and Figure 3A In the atom map whichwas based on the expression of major C atom numbersin various lipid types levels of lipids with carbons and increased whereas those with carbons decreased as compared with healthy control Figure 3BAs compared with the healthy control Figure 4A wenoticed that PI mainly declined in ADC Figure 4B PA 0cZHU of TA B L E carcinoma SCLC as well as lung cancer patients survived or nonsurvivedTop clinical phenomes of patients with adenocarcinoma ADC squamous cell carcinomas SCC or small cell lungPatients with ADCStage at primarydiagnosis ± Stage at recruitmenttime ± TTF1 ± N2 ipsilateralmediastinum ± Napsin A ± Enhanced image ± Location ± N1 ipsilateralparatracheal ± N1 ipsilateral hilum ± CK7Patients with SCCN1 ipsilateral hilum ± Enhanced image ± Stage at recruitmenttime ± Stage at primarydiagnosis ± obscure boundary ± Emphysema ± T tumor ± L1 cycle ± Obstructivepneumoniaatelectasis ± pulmonary nodulePatients with SCLCStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralParatracheal ± T tumor ± N1 ipsilateral hilum ± N2 ipsilateralmediastinum ± Enhanced image ± pulmonary nodule ± N LN ± MaintenancetreatmentPatients survivedStage at primarydiagnosis ± N2 ipsilateralmediastinum ± Enhanced image ± Stage at recruitmenttime ± N1 ipsilateral hilum ± TTF1 ± Napsin A ± Location ± N1 ipsilateralparatracheal ± LobularPatientsnonsurvivedStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralparatracheal ± N2 ipsilateralmediastinum ± N1 ipsilateral hilum ± Enhanced image ± N2 below carina ± TTF1 ± N LN ± T tumor ± ± Abbreviations N degrees of lung cancer metastasis to lymph nodes of TNM category N1 degree that has metastatic lymph nodes near pulmonary center andside of main bronchia N2 degree that has metastatic lymph nodes in the same side of the mediastinum as lung cancer TTF1 thyroid transcription factor1 asan immunohistochemical biomarker for adenocarcinoma CK7 keratin as an immunohistochemical biomarker for epithelial cells L1 cycle number of the firstline chemotherapy cycles ± ± ± in ADC and SCC Figure 4C and lysoPG in SCLC Figure 4D whereas PG and TAG increased in ADC and SCCPE in SCLC and PC in SCC The volcanic map demonstrated the clear patterns of lipid elements significantlyincreased or declined between heathy controls with ADCFigure 4E SCC Figure 4F or SCLC Figure 4G and varied among different subtypes of lung cancerspatient gendersDifferent lipidomics betweenAbout or lipid elements significantly increased and or declined more than twofold in male or femalelung cancer patients as compared with male or femalehealthy controls respectively Tables S4 and S5 Of thosePC and PE mainly elevated in male and female patientswhereas PA declined in both although the number ofPA in male patients was more than in female patientsTable demonstrates genderspecific lipid elements identified by those lipid elements elevated or declined exclusively in either male or female lung cancer patients forexample some of lysoPS PC and PS elevated in malepatients whereas lysoPI and PE in female patients Therewere about or increased or declined lipid elementsdiffered between male and female lung cancer patientsTable 0c of ZHU et alTA B L E adenocarcinoma ADC squamous cell carcinoma SCC or small cell lung cancer SCLC patientsComparisons of clinical phenomes in increased folds and statistical significance Pvalues between each two groups ofTTF1Napsin ABullaeP40HemoptysisEmphysemaSputumCK7HbCoughEGFRVacuole cavityCEAN2 ipsilateral mediastinumNew metastasisP63Cyfra211Obstructive pneumonia atelectasisSmokingPleural pullThirdlineWBCL1 cyclePDL1 tumorPTBronchiectasisPD1 tumorL2 chemo regimenSynMaintenance treatmentNSEN1 ipsilateral ParatrachealCD56CHGT tumorPD1 interstitialSum of all tumors mmN LNKi67Bronchial stenosisN3 opposite sideBurrNeuL2 cyclePulmonary noduleCK56ADC vs SCCFoldsNANANANANANANANAPvaluesNANASCC vs SCLCFoldsNANANANANANANANANAPvaluesNANANAADC vs SCLCFoldsNANANANANANANANAPvaluesNANANANA 0cZHU of Lung cancer subtypeassociated lipid elements significantly elevated or declined alone in patients with adenocarcinomaFoldsPvalues LipidsFolds PvaluesSquamous cell carcinomad181SoC1P240d181S1PPS363TA B L E squamous cell carcinoma or small cell lung cancer more than twofold as compared with healthy control PvaluesSmall cell lung cancerAdenocarcinomaLipidsLipidsElevated twofoldlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPE lysoPG lysoPG lysoPS lysoPS lysoPS PA PA PA PC PC PC 332e PC 161e181PC 352e PC 160e202PC PC lysoPG lysoPI sn1lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PA PA PE PE orFoldsPvaluesPC PC or PC PC PC PC orPE PE Declined twofoldPG PS PS SM240PG PS401PE PE PI PI PI PI PI PI PIP PS PS TAG PA PA PA PA PA PA PA PA PA 0c of ZHU et alF I G U R E Scores of altered lipid elements in variable import in project VIP chart A where top lipid elements were defined amongpatients with adenocarcinoma ADC squamous cell carcinomas SCC small cell lung cancer SCL and healthy controls CON The xaxisrepresents the VIP score and the yaxis represents the lipid elements corresponding to the VIP score The right color grid stands for the relativeconcentration of lipid elements in four groups The degree of altered concentrations increased from green to red The heatmap B describesthe top lipid elements at the high concentration and the degree of lipid elements increased from blue low to brown highF I G U R E less than and respectively as compared with the healthy controlTop six significantly increased lipid elements in patients with ADC A SCC B and SCLC C and stand for the Pvalue 0cZHU of F I G U R E Histography of five component distributions and percentages A measured by partial least squares discrimination analysisPLSDA and the carbon atom map B in healthy controls red and patients with ADC green SCLC orange and SCC blue Each ofselected five principal components represents as the model to interpret that values of abscissa and ordinate represents the distance from thesample nodule to the origin of the center after projecting to a plane in multidimensional space A The atom map describes the expression ofmajor carbon atom number between and in various lipid types BF I G U R E The proportion of main lipid elements of healthy controls A ADC B SCC C and SCLC D and volcanic mapbetween heathy controls with ADC E SCC F or SCLC G respectively The lipid elements were identified on the basis of statistical significance The abscissa represents log values of fold changes where the left side of the first dotted line perpendicular to the abscissa represents fold changes and the right side of the second dotted line represents 2fold changes The vertical coordinate represents “log10 Pvalue Theupper side of the dotted line perpendicular to the ordinate stands for Pvalue less than as compared with healthy controls 0c of ZHU et alFoldsPvaluesPvaluesFemale patients with lung cancerLipidsFoldsTA B L E Genderassociated lipid elements significantly elevated or declined alone in male or female patients with lung cancer morethan twofold as compared with healthy control PvaluesMale patients with lung cancerLipidsElevated twofoldlysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PC PC or PC PC PC 375ePC 160e225180e205PC PC PC PC PC PC PC PC PC PC PC PC PC C1P120 MeanC1P160 MeanC1P240 MeanCer120d171Sod180Sa1Pd181S1Pd181SolysoPC sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS PC PC PE 355p PE 160p204PE 356p PE 160p205PE PE PE 376p PE 180p205 orPE PE PG PG PI 311p PI 160p160PS PS PS PS PS PS Declined twofoldlysoPS PA PA PA PA PA PA PA PA PA 181p204 160e226PE 377p PE 160p226PE PE PE PE orPI PI or or PS PS PA PA Continues 0cZHU et alContinuedTA B L E Male patients with lung cancerLipidsPG PG PS PS PS FoldsPvalues of Female patients with lung cancerLipidsFoldsPvaluesDifferent lipidomics among patientages stags metastases and survival statusAbout and lipid elements significantly elevatedor and declined in lung cancer patients at years old respectively as compared with healthycontrols P We noticed that elements of PG andPS mainly increased in lung cancer patients at all agegroups for example and at 60year group and at to 70year group and and at year group lysoPC and PC increased at 60year group and and at 70year group and PEincreased at to 70year group and at 70yeargroup as detailed in Table S6 Elements of PA mainlydeclined in lung cancer patients at all ages Table S7 Ofthose significantly altered lipid elements and appeared only at 60year to 70year and 70yeargroups respectively and considered as agespecific lipidelements Table LysoPC and lysoPI mainly increasedin 60year and to 70year old patients whereas lysoPEdeclined in 60year group We also compared lipidomicprofiles between patients at early and late stages of lungcancer and found and lipid elements significantlyincreased at early and late stages respectively of whichPE PG ad PS increased in both stages lysoPI in earlystage and PC in late stage Table S8 About and elements declined at early and late stages where the majority was PA Table S9 Table demonstrates stagespecificlipid elements identified by those lipid elements elevatedor declined exclusively at early and late stages of lung cancer for example some of lysoPI and PE elevated at earlystage and lysoPC and PE at late stageWe noticed about or lipid elements significantlyincreased in patients without or with metastasis of whichlysoPI mainly elevated in patients without metastasiswhereas PC and PE in patients with metastasis Table S10The declined number of lipid elements especially PA inpatients with metastasis was significantly higher than inpatients without metastasis Table S11 There were about or elevated or declined lipid elements in patients withmetastasis of which PA was majority of declined elementsin patients with metastasis Table Lipidomic panel also differed between survived andnonsurvived patients There were only eight lipids exclusively elevated in nonsurvived patients that is lysoPS140PC PC PE PE or PE PE PS330 PS372 andPS387 However far more lipids31 elevated alone in survived patients mainly elements of lysoPC lysoPG lysoPIlysoPS and PS On the contrary there were no lipidsdeclined alone in survived patients while lipids in nonsurvived patients of which were PA elements Table clinical phenomes and lipidomesTransomic profiles betweenWe also compared the difference of lipidomic profilesbetween general metabolism statuses of patients indicatedby BMI and between degrees of clinical phenomes measured by DESS scores Levels of lysoPC or lysoPI mainlyelevated in patients with BMI ‰¤ or respectivelyTable S12 whereas the number of declined PA in patientswith BMI ‰¤ was higher than that in patients withBMI Table S13 About BMIassociated lipid elements significantly elevated or declined exclusively inpatients with BMI ‰¤ and about in patients withBMI Table Levels of lysoPC and PE or PG and PSmainly increased in patients with DESS ‰¤ or TableS14 The number of declined PA n in patients withDESS ‰¤ was lower than that in patients with DESS n Figure demonstrates the variation of transomicprofiles among lung cancer subtypes indicated by transomic nodules cross significant networks of clinical phenome and lipidome layersDISCUSSIONThe present study preliminarily found the differencesof lipidomic profiles among patients of different lungcancer subtypes genders ages stages metastatic statusbody qualities and clinical phenome severities Besides itinitially demonstrated clinical phenomeassociated lipid 0c of ZHU et alFolds Pvalues LipidsPatient age Folds Pvalues LipidsPatient age TA B L E Ageassociated lipid elements significantly elevated or declined alone in each age group of patients with lung cancer morethan twofold as compared with healthy control PvaluesPatient age LipidsElevated twofoldC1P120 MeanlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPG d181S1PlysoPC sn1lysoPE190lysoPE191PC PC PC PE 356p PE160p205PE PE PE PE orlysoPG140lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1Folds PvalueslysoPS lysoPS150lysoPS161lysoPS170lysoPS201lysoPS202lysoPS220PA PE PE orPE PE PE PE PE PE orPI 311p PI160p160PI PI PI PI PI361TAG PA PA PG393lysoPS lysoPS lysoPS PC PE PE PG PG PG PS Declined twofoldlysoPE sn1lysoPE sn1lysoPE sn1lysoPE sn2PA PA PS PS PE PE orPE PE PG351PS354PS372PA elements and lipid elementassociated clinical phenomesusing clinical transomics Studies on lipidomic profilesof lung cancer patients have experienced three phasesto detect the difference of lipidomic profiles betweenhealthy and lung cancer patients16 the association ofmultiomics among lung cancer subtypes3 and themolecular mechanism of clinical lipidomicsbased targetlipid elements17 Of those lipidomicsbased data limitedinformation could be adopted to understand the diseaseoccurrence and development phone progression and 0cZHU of Stageassociated lipid elements significantly elevated or declined alone in patients with lung cancer at the early stage or lateFolds Pvalues LipidsPatients at late stageFolds Pvalues LipidsTA B L E stage more than two folds respectively as compared with healthy control PvaluesPatient at early stageLipidsElevated twofoldlysoPE lysoPG lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPS lysoPS lysoPS lysoPS PC PC orlysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS PC PC PE PE PE PE PE 356p PE 160p205PE PE PE PE PE PE PI PI 311p PI 160p160PI PI or or PS PS PC PC PC 375e PC 160e225 or180e205PC PC PC PC PC PC PC PC PC PC PC PC PC PC orPC PC PC PE 355p PE 160p204PE 376p PE 180p205 or181p204 or 160e226PE 377p PE 160p226PE PE PE PE orPE PE PE PE PG PG PG PG PG PG PG PG Patients at late stageFoldsDeclined twofoldlysoPS PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PG PG PS PS PvaluesContinues 0c of ContinuedTA B L E Patient at early stageLipidsPatients at late stageFolds Pvalues LipidsPG PS PS PS ZHU et alPvaluesPatients at late stageFoldsFolds Pvalues Lipidsresponse to therapy due to the lack of link between omicsdata and clinical phenomes Like other omics investigations most genomic data were not tied with clinicalinformation so that with little values to be understoodand applied for clinical precision medicine18 In orderto face the major challenge that most clinical information was descriptive and unmatched with the digitalquantity of omics data clinical phenomes were scoredby DESS and integrated with genomic and proteomicdata of patients with acute respiratory distress syndromeand chronic obstructive pulmonary disease19“ Clinicalphenomes were furthermore integrated with lipidomicprofiles in patients with pulmonary embolism acutepneumonia and acute exacerbation of chronic obstructive pulmonary diseases based on clinical transomicsprinciple15Lipidomic profiles difference between health and lungcancer has been defined and it depends upon methodologies of measurement and analysis sample preparationsand sources and patient populations and status8 Forexample serum levels of lysoPC C260 and C261 and PCC424 and C344 were different between stage I NSCLCand healthy patients22 Some elements and pathwaysof serum PC and PE profiles increased in patients withlung benign disease and earlystage NSCLC as comparedwith healthy whereas few eg PC significantlyelevated in earlystage NSCLC patients alone2 It seemsthat patterns of lipid elements may be associated with thespecificity of lung cancer and stage rather than the intactlipid pathways We performed

" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are “ nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ “] studies haveshown that exosome mirna signaling promotes tumor the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ “] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as œbait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atcc™s instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at —g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturer™s protocol pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatography“mass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm — mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads„¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to “ bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sds“page with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package student™s ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using œthe database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments™ conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p student™s ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p student™s ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p student™s ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [“] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2“ a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn

"These results indicate that FTSJ2 is involved in the inhibition of cancer cell migration and invasion. .0090818.g006 Inhibition of cell migration and invasion upon the over-expression of hFTSJ2 in TE671 cancer cells. (A) The wound healing assay showing that the TE671-hFTSJ2 cells had a reduced migration compared with the untransfected TE671 cells at 12 hours after wounding. (B) Cell migration area at 12 hours after wounding ([healing area/wounding area]—100%). (C) Invasion assay showing that the TE671-hFTSJ2 cells had a reduced invasion compared with the untransfected TE671 cells. The cells that penetrated the Trans-well membrane are shown in purple. (D) Quantity of the cells that invaded the Trans-well membrane ([Giemsa positive area/total area]—100%). The values are equal to?=?the means±SE; n?=?3; *P<0.05 vs. untransfected TE671 cells. Discussion In this study we characterized the mammalian FTSJ2 protein which we presumed to be an ortholog of E. coli RrmJ. RrmJ is known as a 2?-O-ribose MTase which methylates U2552 in the A-loop of the peptidyl transferase center in the 23S rRNA [5]. Um2552 is one of the four 2?-O-methylated nucleotides in rRNA [6]. In a previous study a lack of U2552 methylation has been found to influence the tertiary interactions of U2552 U2555 and C2556; to reduce the conformational dynamics of the A-loop [8]; and to subsequently decrease the ribosome stability and translation efficiency [7] [9] [37]. These results indicate the importance of RrmJ and the methylation of U2552. In our phylogenetic analysis in this study the RrmJ homologs clustered into the following three groups in Eukaryota: FTSJ1/Trm7p FTSJ2/Mrm2p and FTSJ3/Spb1p (). In a comparison of the divergent distance between RrmJ and the ancestral roots of each group FTSJ2/Mrm2p showed the closest relation to RrmJ. In the FTSJ2/Mrm2p protein group S. cerevisiae Mrm2p has been studied extensively. In the mitochondrial rRNA of S. cerevisiae only three nucleotides are modified including the U2791 of the 21S rRNA which is 2?-O-ribose-methylated by Mrm2p. It has been proposed that Mrm2p is the mitochondrial RrmJ ortholog in S. cerevisiae according to the equivalent catalytic positions of Um2791 in the S. cerevisiae mitochondrial 21S rRNA and Um2552 in the E. coli 23S rRNA [6] [12]. However mammalian FTSJ2 remains uncharacterized. Thus we performed a sequence alignment of human FTSJ2 with RrmJ Mrm2p and FtsJ2 in M. jannaschii and three typological invertebrate species () and we compared the 3D structures of RrmJ human FTSJ2 and porcine FTSJ2 (Figure S1). A previous study showed the highly conserved catalytic tetrad K-D-K-E in site-specific 2?-O-ribose MTases [10] and this catalytic tetrad was also present in our sequence alignment. Furthermore a sequence alignment revealed the highly conserved amino acids involved in SAM binding. However interestingly the 3D structure of human FTSJ2 showed a different orientation for the first residue of the catalytic tetrad (lysine) compared with RrmJ. This difference may indicate a different A-loop structure of the rRNA substrate or a different catalytic mechanism of the FTSJ2/Mrm2p protein in mammals. Because S. cerevisiae Mrm2p is localized in the mitochondria [12] we hypothesized that hFTSJ2 is a mitochondrial protein. We used immunofluorescence and Western blot analysis to verify that hFTSJ2 was predominantly located in the mitochondria but not in the nucleus or cytoplasm (Figures 3C and 3D). In addition E. coli RrmJ is well known as a heat shock protein. The rrmJ mRNA expression increases over 20-fold after heat shock [3] and the rrmJ deletion strain fails to adapt to heat shock temperatures [7]. In S. cerevisiae the growth of the mrm2 deletion strain at 37°C is slightly reduced on glucose-containing medium and severely reduced on glycerol-containing medium [12]. Thus to evaluate the heat shock response of the RrmJ ortholog in mammals we tested the heat shock response of piglets at 30°C or 35°C. The large intestine lung and bladder showed an up-regulated expression of Ftsj2 mRNA at temperatures of 30°C and 35°C but only the lung tissue demonstrated a simultaneous heat shock response with the up-regulation of Hsp70.2 mRNA (). This finding in the lung may have been caused by the direct exposure of this tissue to the increased temperature through the inhalation of hot air. However under these heat shock treatments for the piglets only 5 (small intestine muscle lung kidney and liver) of the 11 tissues showed an up-regulation tendency of Hsp70.2 expression possibly because of the systemic effect of the response of the warm-blooded piglets to the heat shock stress. Furthermore to eliminate this systemic effect and to confirm the FTSJ2 mRNA up-regulation in the lung a human lung adenocarcinoma cell line (A549) was subjected to heat shock for 1 hour and allowed to recover at 37°C. The results of this experiment showed a 1.5-fold increase in the hFTSJ2 expression at both 42°C and 45°C and then a gradual return to its normal level after the recovery period (). Although the exact role of FTSJ2 in the heat shock response in mammals is unknown these results indicate that FTSJ2 inherited the HSP characteristics of its orthologs in E. coli and S. cerevisiae. In the previous studies of HSPs such as HSP70 and HSP90 it has been demonstrated that the heat shock responses are highly conserved during evolution. From Prokaryota to Eukaryota and Protozoa to Metazoa the HSPs represented the universal protein structures and similar physiological functions and following evolution the HSPs diverged and translocated into different anelles [1] [2] [38]“[40]. These characteristics are in alignment with the results of the RrmJ phylogenic analysis and the conservation of the heat shock response properties in mammals. In addition to certain small HSPs (i.e. HSP32 HSP25 and HSP22) most of the HSPs are expressed in all types of tissues [2] [41] and our results showed that Ftsj2 was expressed in all of the 13 normal piglet tissues (Figure S2). These results indicated that FTSJ2 was not only involved in the heat shock response but also might be necessary for mitochondrial functions under normal condition according to the mitochondrial localization of FTSJ2. In addition previous functional studies have shown that the HSPs (i.e. HSP70 and HSP90) are involved in tumorigenesis and the inhibition of apoptosis in cancer cells [40] [42]“[44]. Similarly the amplification of the genetic locus of FTSJ2 has been discovered in several NSCLC clinical samples and was considered as a novel oncogenic locus [20]. In our study the expression of FTSJ2 was also shown in different cancer cells (hepatocarcinoma lung adenocarcinoma and rhabdomyosarcoma cells). However in the human lung adenocarcinoma cell sublines CL1-0 and CL1-5 [45] we found that hFTSJ2 mRNA was decreased in the more invasive CL1-5 cells compared with the less invasive CL1-0 cells. Moreover the TE671-hFTSJ2 cells which over-expressed the hFTSJ2 protein showed a decrease in cell migration and invasion (). These results indicate that the mitochondrial hFTSJ2 protein exhibits an additional function to suppress cancer cell metastasis. Previous reports have suggested that hFTSJ2 functions in the mitochondria. Thus it is reasonable that FTSJ2 is required for extensive ATP production through respiration in the mitochondria of proliferating cancer cells [46]“[48]. In contrast according to recent studies a mitochondrial complex I and NAD+/NADH imbalance enhances the metastasis of breast cancer cell lines [49] and the dynamics of mitochondrial fusion or fission also regulates cell migration and invasion [50] [51]. These results indicate that the invasiveness of cells is affected by the condition and state of their mitochondria. In we characterized FTSJ2 as an ortholog of the E. coli 23S rRNA 2?-O-ribose MTase and showed that it functions in the mitochondria. We also provided evidence that FTSJ2 is a novel heat shock protein that is over-expressed after heat shock treatment in both piglet lung and lung adenocarcinoma cells. Surprisingly FTSJ2 may also be involved in the inhibition of cancer cell migration and invasion by influencing the mitochondrial functions. Accession Numbers The GenBank accession numbers of the protein sequences which were used in the phylogenetic tree construction and the protein sequences alignment are labeled in and . The protein coding regions of the porcine Ftsj1 and Ftsj2 mRNA were first sequenced in this study and the GenBank accession numbers of the corresponding porcine Ftsj1 and Ftsj2 mRNA are EU694401 and EU694400 respectively and the porcine FTSJ1 and FTSJ2 proteins are ACH57153 and ACH57152 respectively. Supporting Information Figure S1 Three-dimensional protein structures of E. coli RrmJ human FTSJ2 and porcine FTSJ2. (A) The protein structure of E. coli RrmJ which was resolved by B¼gl et al. (2000) (PDB ID: 1EIZ) [7]. (B) The protein structure of human FTSJ2 which was resolved by Wu et al. (2009) (PDB ID: 2NYU) [36]. (C) The protein structure of porcine FTSJ2 which was predicted using the SWISS-MODEL website with human FTSJ2 as a template. The ?-helices and ?-strands are shown in green and yellow respectively. The SAM residues and the K-D-K-E catalytic center are shown in the ball and stick representations respectively. (TIF) Click here for additional data file. Figure S2 Porcine Ftsj2 mRNA expression in porcine tissues. (A) Expression of porcine Ftsj2 mRNA as measured by semi-quantitative RT-PCR. Porcine ?-actin mRNA was used as a loading control. (B) Quantification of the porcine Ftsj2 mRNA expression which normalized to the ?-actin mRNA expression. The values are equal to?=?the means of duplicate experiments. (TIF) Click here for additional data file. The authors would like to thank Dr. Jeremy J.W. Chen for providing the CL1-0 and CL1-5 cell lines. We also like to thank our colleagues (Drs. Tung-Chou Tsai Yu-Tang Tung and Zi-Lun Lai) in the Molecular Embryology and DNA Methylation Laboratory for their help with discussions and technical issues. References 1 AngM LiberekK SkowyraD ZyliczM GeopoulosC (1991) Biological role and regulation of the universally conserved heat shock proteins. J Biol Chem266: 24233“242361761528 2 BenjaminIJ McMillanDR (1998) Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. 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Oncogene33: 567“57723318458 49 SantidrianAF Matsuno-YagiA RitlandM SeoBB LeBoeufSE et al (2013) Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. J Clin Invest123: 1068“108123426180 50 LinCS LeeHT LeeSY ShenYA WangLS et al (2012) High mitochondrial DNA copy number and bioenergetic function are associated with tumor invasion of esophageal squamous cell carcinoma cell lines. Int J Mol Sci13: 11228“1124623109849 51 ZhaoJ ZhangJ YuM XieY HuangY et al (2013) Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Oncogene32: 4814“482423128392 J Thorac Oncol J Thorac Oncol JTO Journal of Thoracic Oncology 1556-0864 1556-1380 Lippincott Williams & Wilkins 24419415 4132025 00009 10.1097/JTO.0000000000000048 Original Articles Non-Small Cell Lung Cancer Effectiveness of Gefitinib against Non“Small-Cell Lung Cancer with the Uncommon EGFR Mutations G719X and L861Q Watanabe Satoshi MD PhD * Minegishi Yuji MD PhD   Yoshizawa Hirohisa MD PhD * Maemondo Makoto MD PhD ¡ Inoue Akira MD PhD § Sugawara Shunichi MD PhD ? Isobe Hiroshi MD PhD ¶ Harada Masao MD PhD # Ishii Yoshiki MD PhD ** Gemma Akihiko MD PhD   Hagiwara Koichi MD PhD    Kobayashi Kunihiko MD PhD ¡¡ *Bioscience Medical Research Center Niigata University Medical and Dental Hospital Niigata Japan;  Department of Internal Medicine Division of Pulmonary Medicine Infections Disease and Oncology Nippon Medical School Tokyo Japan; ¡Department of Respiratory Medicine Miyagi Cancer Center Miyagi Japan; §Department of Respiratory Medicine Tohoku University Sendai Japan; ?Department of Pulmonary Medicine Sendai Kousei Hospital Sendai Japan; ¶Department of Medical Oncology KKR Sapporo Medical Center Sapporo Japan; #Department of Respiratory Medicine National Hospital anization Hokkaido Cancer Center Sapporo Japan; **Department of Pulmonary Medicine and Clinical Immunology Dokkyo Medical University School of Medicine Mibu Japan;   Department of Respiratory Medicine Saitama Medical University Saitama Japan; and ¡¡Department of Respiratory Medicine Saitama International Medical Center Saitama Japan. Address for correspondence: Hirohisa Yoshizawa MD PhD Bioscience Medical Research Center Niigata University Medical and Dental Hospital 1“754 Asahimachi-dori Niigata 951“8510 Japan. E-mail: nnys@med.niigata-u.ac.jp 2 2014 23 1 2014 9 2 189 194 Copyright © 2013 by the International Association for the Study of Lung Cancer 2013 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivitives 3.0 License where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. Introduction: In non“small-cell lung cancer an exon 19 deletion and an L858R point mutation in the epidermal growth factor receptor (EGFR) are predictors of a response to EGFR-tyrosine kinase inhibitors. However it is uncertain whether other uncommon EGFR mutations are associated with sensitivity to EGFR-tyrosine kinase inhibitors. Methods: A post-hoc analysis to assess prognostic factors was performed with the use of patients with EGFR mutations (exon 19 deletion L858R G719X and L861Q) who were treated with gefitinib in the NEJ002 study which compared gefitinib with carboplatin-paclitaxel as the first-line therapy. Results: In the NEJ002 study 225 patients with EGFR mutations received gefitinib at any treatment line. The Cox proportional hazards model indicated that performance status response to chemotherapy response to gefitinib and mutation types were significant prognostic factors. Overall survival (OS) was significantly shorter among patients with uncommon EGFR mutations (G719X or L861Q) compared with OS of those with common EGFR mutations (12 versus 28.4 months; p = 0.002). In the gefitinib group (n = 114) patients with uncommon EGFR mutations had a significantly shorter OS (11.9 versus 29.3 months; p < 0.001). By contrast OS was similar between patients with uncommon mutations and those with common mutations in the carboplatin-paclitaxel group (n = 111; 22.8 versus 28 months; p = 0.358). Conclusions: The post-hoc analyses clearly demonstrated shorter survival for gefitinib-treated patients with uncommon EGFR mutations compared with the survival of those with common mutations and suggest that the first-line chemotherapy may be relatively effective for non“small-cell lung cancer with uncommon EGFR mutations. Gefitinib G719X L861Q NEJ002 Uncommon epidermal growth factor receptor mutations OPEN-ACCESS TRUE The clinical efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib has been demonstrated in non“small-cell lung cancer (NSCLC) patients in whom standard chemotherapy has failed.12 Further studies have revealed that the presence of activating mutations in the EGFR kinase domain is strongly associated with the therapeutic efficacy of EGFR-TKIs.34 Randomized phase 3 trials have demonstrated that EGFR-TKIs significantly improve median progression-free survival (PFS) compared with platinum-doublet therapy in EGFR-mutated patients.5“8 However not all mutations in the EGFR kinase domain are responsive to EGFR-TKI treatment. These phase 3 trials have shown that EGFR-TKIs are effective for patients with common EGFR mutations such as an exon 19 deletion or the L858R point mutation which account for more than 90% of EGFR mutations. Retrospective studies and case reports suggest that some uncommon mutations are associated with sensitivity to EGFR-TKIs.9“20 These mutations include G719X in exon 18 which accounts for approximately 3% of EGFR mutations and L861Q in exon 21 which represents approximately 2% of EGFR mutations. However these uncommon EGFR mutations have not been clearly shown to be predictive markers for the efficacy of EGFR-TKIs because of their low frequency. To investigate the efficacy of gefitinib in patients with uncommon mutations we conducted a post-hoc analysis of the NEJ002 which compared gefitinib and carboplatin-paclitaxel as first-line therapies for advanced NSCLC with activating EGFR mutations. PATIENTS AND METHODS Patient Population We retrospectively analyzed the data of 225 patients who received gefitinib treatment at any point in the NEJ002 study.6 The eligibility criteria of the NEJ002 study included the presence of advanced NSCLC harboring an EGFR mutation (exon 19 deletion or L858R G719X or L861Q point mutation) without the resistant EGFR mutation T790M (identified using the peptide nucleic acid“locked nucleic acid polymerase chain reaction clamp method) no history of chemotherapy an age of 75 years or younger a performance status of 0 to 1 and appropriate an function.2122 Patients provided a written informed consent. The study was conducted in accordance with the Helsinki Declaration of the World Medical Association. The protocol was approved by the institutional review board of each participating institution. Treatment Eligible patients were randomly assigned to receive either gefitinib (250 mg/day)"

" tumor mutational burden tmb has both prognostic value in resected nonsmall cell lung cancernsclc patients and predictive value for immunotherapy response however tmb evaluation by wholeexomesequencing wes is expensive and timeconsuming hampering its application in clinical practice in our study weaimed to construct a mutational burden estimation model with a small set of genes that could precisely estimatewestmb and at the same time has prognostic and predictive value for nsclc patientsmethods tmb estimation model was trained based on genomic data from nsclc samples from the cancergenome atlas tcga validation was performed using three independent cohorts including rizvi cohort and ourown asian cohorts including earlystage and n latestage asian nsclc patients respectively tcga data wereobtained on september the two asian cohort studies were performed from september to march pearson™s correlation coefficient was used to assess the performance of estimated tmb with westmb thekaplanmeier survival analysis was applied to evaluate the association of estimated tmb with diseasefree survivaldfs overall survival os and response to antiprogrammed death1 pd1 and antiprogrammed deathligand pdl1 therapyresults the estimation model consisted of only genes correlated well with westmb both in the training setof tcga cohort and validation set of rizvi cohort and our own asian cohort estimated tmb by the 23gene panelwas significantly associated with dfs and os in patients with earlystage nsclc and could serve as a predictivebiomarker for antipd1 and antipdl1 treatment responsecontinued on next page correspondence zhangli6mailsysueducn jie_969163comzlhuxi163com yanhua tian jiachen xu and qian chu contributed equally to this work5state key laboratory of oncology in south china collaborative innovationcenter for cancer medicine sun yatsen university cancer center eastdong feng road guangzhou guangdong china1state key laboratory of molecular oncology department of medicaloncology national cancer centernational clinical research center forcancercancer hospital chinese academy of medical sciences and pekingunion medical college panjiayuan south lane chaoyang districtbeijing chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctian bmc medicine page of continued from previous pages the 23gene panel instead of wes or the currently used panelbased methods could be used toassess the westmb with a high relevance this customized targeted sequencing panel could be easily applied intoclinical practice to predict the immunotherapy response and prognosis of nsclckeywords tmb estimation 23gene panel prognostic and predictive value nonsmall cell lung cancertoimmuneinhibitorscheckpoint tumor mutational burden tmb commonly defined asthe number of nonsynonymous mutations has been proposed as a promising predictive biomarker for the responseicisimportantly this metric tightly correlates with overallsurvival os in resected nonsmall celllung cancernsclc patients in rizvi demonstratedthat an increased number of nonsynonymous mutationswere associated with improved objective response durable clinical benefit dcb and progressionfree survivalpfs in nsclc patients who received antiprogrameddeath pd1 therapy clinical studies have also revealed a significant correlation between tmb and objective response rate orr to icis in multiple tumortypes [“] in addition devarakonda recently reported that high tmb was associated with a better survival prognosis in patients with resected nsclc andthe benefit of adjuvant chemotherapy was more pronounced in patients with low tmb the gold standards for tmb calculation are throughwholegenome sequencing wgs or wholeexome sequencing wes however several obstacles such as thehigh demand for quality and quantity of tissue samplesthe cost and time consumption and the unavailabilityfor translation to tmb evaluation by circulating tumordna ctdna in blood btmb hinder the clinicalapplication of these techniques as a result targetednext generation sequencing ngs of cancerrelatedgene panels cgp has been developed serving as surrogates for wes for tmb estimation to date the foodand drug administration fda has approved severalngs panels for tmb estimation eg foundationonecdx f1cdx and memorial sloan kettering cancercenter™s integrated mutation profiling of actionablecancer targets mskimpact which include about“ genes and cover over one megabase of codingdna [ ] recently many new ngs panels consistingof different numbers of genes have been developed andvalidated most of which were designed initially for guiding the use of target therapies these panels mainly include cancerrelated oncogenes and tumor suppressenes many of which do not contribute to or even negatively correlate with tmb thus are not accurate fortmb evaluation besides inclusion of these genes in anngs panel enlarges the panel size used for tmbis importantestimation and can lead to an inferior costeffective consequence itto note that cancer typespecific mutation load estimation models have proven tobe necessary because of the different mutation landscapes among varying tumor types although dnadamage repair ddr genes negatively predictive genesstk11 and keap1 and tmbassociated genes such asmuc16 pole pold1 and ttn have been included inthe ngs panels for tmb evaluation [“] with theburgeoning developments in immunotherapy there is aneed for more specific panels that focus on tmb estimation for nsclcherein by using the cancer genome atlas tcgadatabase as a training set and multiple realworld cohorts as a validation set we constructed an optimizedtmb estimation model with the smallest number ofcarefully selected tmbassociated genes that could beused as both predictive markers for immunotherapy andprognosis biomarkers for resected nsclc patientsmethodspatient cohortsgenomic and clinical data for nsclc samples including lung adenocarcinoma luad and lungsquamous cell carcinoma lusc samples were downloaded from tcga database for the model constructionfor the validation of the model three independent cohorts were used including a previously published studythe rizvi cohort a surgery cohort composing of earlystage nsclc patients who underwent surgicaltreatment and a zs immunotherapy cohort composingof advanced nsclc patients who received ici treatment all the patients in the zs immunotherapy coreceived either antipd1 nivolumab n hortpembrolizumab n shr1210 n or antipdl1 atezolizumab n monotherapy agents there are patients who received durable clinical benefit dcbantipd1 n antipdl1 n and patientswith no durable benefit ndb antipd1 n antipdl1 n all three validation cohorts were used toevaluate the performance of the tmb estimation modeladditionally the surgery cohort was also used for survival validation in resected nsclc patients both therizvi and immunotherapy cohorts were also used forvalidation of ici outcome predictability in advancednsclc patients the clinical details for all enrolled 0ctian bmc medicine page of patients were collected the treatment efficacy for thosetreated with immunotherapy was assessed using response evaluation criteria in solid tumors recistversion with durable clinical benefit dcb definedas partial or stable disease lasting over months allprocedures were approved by the ethics committees ofthe national cancer center all patients provided written informed consentwholeexome sequencing and data processingwe performed wholeexome sequencing of samplesfrom two cohorts in the validation setincluding earlystage nsclc patients who underwent surgicaltreatment and advanced nsclc patients who received ici treatment for those earlystage nsclcpatients both tumor and matched normal samples wereobtained and subjected to wes briefly dna librarieswere prepared using the mgieasy exome capture v4probe set capture kit cat no with a capture region size of mb bgiseq instruments wereused for pairend sequencing × bp the data wereprocessed according to the manufacturer™s protocol the mean coverage was × and × in tumor andnormal samples respectivelyfor those advanced nsclc patients biopsy specimens were available for wes the genomic dna wasextracted using the qiaamp dna ffpe tissue kit andquantified using the dsdna hs assay kit thermofisher scientific usa libraries were constructed withthe kapa hyper prep kit kapa biosystems usa anillumina hiseq4000 platform was used for sequencingwith pe150 sequencing chemistry illumina usa the average coverage depth was ×candidate gene selectiongenomic data for nsclc samples from tcgawere used for candidate gene selection which were usedto construct the mutation load estimation model thecandidate genes were selected based on two criteria mutation frequency higher than or equal to and significant association with mutation load the mutationfrequency of a gene was calculated as the percentage ofpatients with mutation in the gene mutation loadassociated genes were defined as where the westmbwas significantly different between the patients with themutated gene and those with wildtype counterpartsadditional file table s1mutation estimation model constructionthe mutation estimation model construction was basedon tcga data in the training set in detail the first stepwas to build a mutation estimation model using thefewest genes which tightly associated with westmbin our study we constructed the estimation model bysimply randomly selecting a specified number of genesfrom allthe genes or tmbassociated genes andsummed the mutational number as the estimated tmbunder every given number of genes the procedure wasrepeated times resulting in random modelswe then calculated the pearson correlation coefficientr between the estimated and actual mutation load ofwestmb the results allowed us to select the modelwith highest r under the specified number of genes thenext step was to identify which of those best modelsunder the specified number of genes correlated with theclinical outcomes of overall survival os and diseasefree survival dfs the final step was to select a modelusing the fewest genes that tightly associate with thewestmb and have both prognostic value for thoseearlystage nsclc patients and predictive value forthose latestage nsclc patients who received icitreatmentrna expression difference between tmb high and lowgroupsto compare gene expression patterns we downloadedan mrna data set of nsclc patients from tcgadatabase mrna expression was analyzed using gene setenrichment analysis gsea httpsoftwarebroadinstitutegseaindexjsp we divided these patientsinto estimated high ‰¥ mutational counts and lowtmb groups mutational counts and identifiedwhether immunerelated gene signatures associated withtumor mutation status the genes found to be on theleading edge of the enrichment profile were subjected topathway analysis genes with expression over in morethan of the samples were included in the gseathe normalized enrichment score nes is generally theprimary statistic for examining gene set enrichmentresultsstatistical analysisthe mannwhitney u test was used to assess thedifferences in the mutation load between the twogroups the genes with kruskalwalliscorrected pvalues lower than were identified as the mutationloadassociated genes and selected as potential candidate genes survival analysis was performed using thekaplanmeier curves with a p value determined by alogrank test and the statistical tests were twosidedand considered statistically significant at p unless otherwise stated the analyses were performedusing graphpad prism version graphpad prismcorrelations between estimated mutation burden andwholeexome sequencingcalculated tmb were determinedcorrelation coefficient theanalyses were performed using r353by pearson™s 0ctian bmc medicine page of resultscandidate gene selection for model constructionthe flowchart of the construction of estimation model isshown in fig s1 in additional file the somatic mutation data of cases of nsclc were downloaded fromtcga database as the training set tcga cohort including adenocarcinoma and squamous cell carcinoma subtypes of nsclc additional file table s2subsequently a mutation matrix including screened nonsynonymous mutations in genes was generatedfurthermore we identified genetic alterations in genes with mutation frequency ‰¥ in general nsclcpatients and significantly correlating with westmb pvalue range 695eˆ’ to 452eˆ’ these genes werethen used as candidate genes for the construction of thetmb estimation model additional file table s3construction of the tmb estimation modelgenes used for the tmb estimation model were randomly selected from the candidate genes and theserialrandom models theestimated tmb was defined as the sum of all nonsynonymous mutation counts of the selected genes undereach specified number of abstracted genes the procedure was repeated times thus resulting in separatecorrelations ofestimated tmb by these random models and westmbwere evaluated using the pearson correlation coefficientr as expected the correlations between the estimationmodels and westmb increased with the number ofgenes fig 1a b additional file fig s2a b compared with unselected genes in the range of genomicgenes the estimated tmb based on selected geneswas significantly more closely associated with westmb in terms of either the mean or the maximum rfig 1c d additional file fig s2c d the maximumr increased from with one gene included to greaterthan with genes included and then reached aplateau when the included gene number exceeded the r values were comparable though increased slowlyas the number increased fig 1b we asserted that rfig the correlation of westmb and tmb as estimated by different gene panels a b correlation is represented by the pearson correlationcoefficient r genes used for the mutation model construction were either from unselected genes a or from selected genes b that correlatewith westmb c d comparisons of mean c and maximum d r of estimated tmb and westmb using unselected genes or selected genes 0ctian bmc medicine page of greater than in the estimation models was acceptable as such we considered a model with this effectbut including the least number of genes an ideal modelfor clinical applicationin reference to previous reports that tmb is associated with prognosis in patients with resected nsclcsthe optimal tmb estimation model was further evaluated based on the correlation of estimated tmb with osand dfs in models with r over ultimately we constructed an estimated tmb model with only genesand r of p fig 2a additional file which was significantly associated with both os anddfs fig 2b c the cutoff value of the estimated tmbby the 23gene panel was defined as mutational countsthe median value of estimated tmb based on tcgadatabase additional file fig s3a b that were equalor over mutational counts as tmbhigh cases and lessthan mutational counts as tmblow ones these genesincluded unc13c hmcn1 znf536 kmt2d ush2axirp2 pcdh15 ahnak2 adgrl3 reln nf1 ttnadgrg4 cubn cacna1e mrc1 col11a1 nav3csmd1 apob csmd3 col22a1and epha5additional file table s4 the model yielded goodperformances in both subtypes of nsclc with correlations of for luad additional file fig s4aand for lusc additional file fig s4b theaverage cds length of these genes was 12k nucleotides 3k“80k additional file table s4 and the totallength was 028m nucleotides which was considered tobe a great reduction of sequencing cost for mutationload estimation we concluded that the 23gene panel isthe ideal model based on tcga training setanalytic validation of the 23gene panel in asian resectednsclc patientsto validate the performance of the estimation model weconducted wes on chinese stage ia“iiia nsclcpatients after radical pneumonectomy surgery cohortadditional file table s1 the correlation of 23genetmb with wes was r p fig 3a asshown in fig 3b tmbhigh ‰¥ mutational counts according to the 23gene panel associated with a betterdfs compared with those with tmblow logrank p besides a tendency towards improved os wasobserved in the patients with higher estimated tmbthough a statistical difference was not reached due tothe fact that most patients were still alive fig 3cperformance verification by comparing the 23gene panelwith other commercial panelsnext we compared the 23gene panel with two commercial panels based the earlystage nsclc data including f1cdx genes and mskimpact genes there are two overlap genes between the gene panel with f1cdx and mskimpact namelynf1 and epha5 the 23gene tmb has a tight correlation with the tmb estimated by f1cdx f1cdxtmbor mskimpact msktmb r and respectively both p fig 4a b in additionwhen the genes were added to the two commercialpanels the correlation of the incorporated panels withwestmb significantly increased from ci“ to ci “ p for f1cdx fig 4c d and from ci“ to ci “ p for mskimpact fig 4e f to further verify thespecificity of these 23gene panels we compared themwith other randomly selected gene panels from the genes the procedure was repeated timesresulting in the random pearson correlation coefficientsfrom to of f1cdx plus random genesand from to of msk plus random genes the performance of our 23gene model was better than of random models which indicated the irreplaceability of these genesfig tmb estimation model construction based on tcga data in the training set a the correlation of 23gene tmb with westmb is with an empirical p value of r of p b the overall survival is significantly higher in the tmbhigh group ‰¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the diseasefree survival is significantly higher in thetmbhigh group than in the tmblow group with logrank test p 0ctian bmc medicine page of fig validation of the tmb estimation model based on the earlystage nsclc patients in the validation set a the pearson correlationcoefficient of estimated tmb by the 23gene panel and westmb is with an empirical p value of r of p b the diseasefree survivalis higher in the estimated tmbhigh group ‰¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the overall survival is comparable in the two groups with logrank test p fig performance evaluation of the 23gene panel against commercially used gene panels a b the pearson correlation coefficient of 23genetmb with f1cdxtmb a and msktmb b c d the pearson correlation coefficient of westmb with f1cdxtmb c and incorporated panel of cancerassociated genes in f1cdx with 23gene panel f1cdx genetmb d e f the pearson correlation coefficient of westmb withmsktmb e and incorporated panel of cancerassociated genes in mskimpact with genepanel msk genetmb f 0ctian bmc medicine page of f1cdxbased on the survival data from our earlystagensclc patients significant correlations were observedbetween survival outcomes dfs and the tmb levelstratified withor mskimpact paneladditional file fig s5a c interestingly the genescould improve the association of these two commercialpanels with dfs additional file fig s5b d if the incorporated panels were used for analysis tmbhigh estimated by both of the two new panels f1cdx 23genepanel or mskimpact 23gene panel demonstratedimproved dfs compared with those of estimated tmblow under the cutoff values indicated in fig s3c and s5of additional file immuneregulatory gene expression signatures stratifiedby tmb level based on the 23gene panelto investigate the difference in immune status betweentmbhigh and tmblow estimated by the 23genepanel we analyzed immuneregulatory gene expressionsignatures based on the rnaseq data of nsclccases from tcga the gsea revealed a prominent enrichment of mrna signatures involved in the inflammainterferonα γ ifnα γtoryresponse tnfαresponse il6jakstat3 signaling and allograft rejection fig immunotherapy response prediction by the established23gene panelfinally we analyzed the performance of tmb estimatedby the 23gene panel in the prediction of response toicis using two independent nsclc cohorts in therizvi cohort the correlation between the tmb estimatedby the 23gene panel and wes was empirical pvalue of r fig 6a the estimated tmb was significantly different between the patients with durableclinical benefit dcb a partial or stable response lastingover months and no durable benefit ndb mannwhitney p fig 6b survival analysis was thenapplied for the comparison of the pfs between the patients n with tmbhigh ‰¥ counts and tmblow counts by the 23gene panel patients withtmbhigh demonstrated significantly improved pfscompared with those with tmblow vs months logrank p fig 6cto further validate the performance of the estimationmodel for response to icis we performed wes of fig gene expression differences between the estimated high tmb and low tmb groups a“f tmbassociated pathways such as inflammatoryresponse tnfα signaling via nfκb interferon α response il6jakstat3 signaling interferon γ response and allograft rejection nes normalizedenrichment score fdr false discovery rate 0ctian bmc medicine page of fig immunotherapy response estimation by the 23gene panel a the correlation of the estimated tmb with westmb using the rizvi datan b estimated tmb in tumors from patients with dcb n or with ndb n mannwhitney p c pfs in tumors withestimated tmbhigh n compared to tumors with tmblow n in patients in the rizvi cohort hr ci to logrank p d the correlation of estimated tmb with westmb using the latestage nsclc patient cohort n e estimated tmb in tumors frompatients with dcb n or with ndb n mannwhitney p f pfs in tumors with estimated tmbhigh n compared totumors with tmblow n in patients in the latestage nsclc patient cohort hr ci to logrank p advanced stage iiib“iv nsclcs in another asian cohort zs immunotherapy cohort all of these patients received with antipd1 or antipdl1 treatmentthe r between the estimated and actual mutation burden was calculated to be empirical p value of r fig 6d the estimated tmb was significantlydifferent between the patients with dcb and ndbmannwhitney p fig 6e the pfs was associated with estimated tmb logrank p fig 6fdemonstrating that the estimated mutation burden derived from caucasian nsclcs from tcga could predict the immunotherapy treatment response quite wellin asian patients we further calculated the hr at different cutoff values in the zs immunotherapy cohort andfound the mutational counts in this cohort resultedthe best hr value additional file fig s6 as a resultwhen applied in clinical practice the cutoff value stillneeds to be further evaluated accordinglycomparison of the 23gene panel with previouslyreported tmbrelated genesmutations in ttn muc16 pole and pold1 havebeen previously reported to correlate with elevated tmblevels [“] the frequencies ofthese genes innsclc based on cases from tcga were and respectively westmb was significantly different between the patients with these mutatedandthose with wildtypegenescounterpartsadditional file fig s7 however only muc16 mutations exhibit significant correlation with os and dfs intcga cohort additional file fig s7ac while theyfailed to confirm the results in our surgery cohortadditional file fig s8 notably none of these genemutations could predict the response or pfs in eitherthe rizvicohortadditional file fig s9cohort or ourimmunotherapydiscussionin the present study we developed a novel and optimaltmb estimation model composed of only geneswhich allowed precise estimation ofthe wesbasedtmb both in earlystage and latestage nsclc patientsimportantly our established 23gene panel can successfully predict the survival outcomes in both resectednsclcs and patients receiving icis in multiple validation cohorts to the best of our knowledge our tmbestimation model is both the first and the smallest paneldescribed to date which can be used as a biomarker tostratify patients not only after radical pneumonectomybut also with advanced nsclc receiving icisthe total cds length of the 23gene panel was 028mnucleotides with an average of 12k 3k“80k the ttnis also included in our panel although it has the longestcds length of 81k the total length was acceptable when 0ctian bmc medicine page of ttn is included besides in a recent study ttn mutation was reported to be associated with tmb in solid tumors including nsclc and correlated with response toicis as a result the 23gene panel was consideredto be a great reduction of sequencing cost for mutationload estimationseveral cancerrelated genes have been previously reported to be associated with westmb in some cancertypes for example melanoma patients with lrp1b mutations exhibited a higher mutationalload than thosewith the wildtype gene li reported that mutations in muc16 are associated with tmb and survivaloutcomes in patients with gastric cancer twoddrrelated genes pole and pold1 were also shownto correlate well with westmb in pancancer types undoubtedly it would be ideal to utilize a singlegene to estimate tmb and effectively predict responseto immunotherapy however we found that singly allthese genes failed to correlate well with the efficacy oficis or survival outcomes after resection the correlationof any individual gene with westmb was moderatemean r “ these results indicate thatusing a single gene to estimate tmb is insufficienttheoretically the larger a ngs gene panel the closerthe estimated tmb is to the actual amount howeverthe costeffective balance for clinical usage must be considered in particular when tmb is detected using peripheral blood super sequencing depth eg “× due to the low abundance of circulating tumordna will significantly drive up the cost to datetwo commercial gene panels f1cdx and mskimpact have been widely used for tmb estimation thesetwo panels demonstrate good performance in correlationwith westmb our established gene panel whichincludes a very limited number of genes demonstratedcomparable correlation coefficients with these two largepanelsindicating the promising reliability of a smallpanel as a surrogate for westmb notably the majority of genes used in our model were not included in thecurrently used commercial gene panels if the genes inour panel were incorporated into the big commercialgene panels the correlation coefficients with westmbincreased these results demonstrate that the geneswe have selected here may be used independently or ascomplement to the currently used gene panels specificfor nsclc inclusion of the genes should be considered in future ngs gene panelsrecently lyu developed a small gene panel with genes to estimate actual tmb derived from luads in tcga database the construction and validation cohorts used for lyu ™s 24gene panel weremainly from caucasian patients however our 23genepanel though also derived from tcga database wassuccessfully validated in multiple asian patient cohortsthese results suggest that our 23gene panel may bemore suitable to nsclc and applicably potent regardless of race and subtypes additional file fig s10similar with the findings of devarakonda weobserved that high tmb associated with improved os inresected nsclc patients in colon cancer patients withresected stage ii mismatch repair deficiency high tmbhas been utilized as a good prognostic biomarker indeed these results possess internal rationality both highneoepitope burden and intense til infiltration have been associated with favorable survival outcomes inearlystage lung cancer high tmb may reflect the immunogenicity in some degree which could mediate theshaping of tumorhost immune interactions taken together these and our findings suggest that quantifyinggenomic instability through tmb estimation can be usedto stratify patients so as to guide adjuvant treatmentowing to the lack of information on hlai it is difficultto judge whether the predictive value of our gene panel isdue to neoantigen generation derived from the includedgene mutations or if the estimated tmb based on the gene panel is simply a representative reflection of genomicinstability as an œaccompanying passenger the otherlimitation of our study is the small number of patientswho received the immunotherapy treatment thus a larger number of cases from a multicenter study are requiredfor the validation of the performance of the treatment response prediction in addition our validation cohorts wereretrospective a prospective study is necessary to translateour estimation model into clinical practice in addition totmb other features such as pdl1 expression microsatellite instability and neoantigen burden have emerged aspotential predictive biomarkers for icis [“] howeverchallenges in defining cutoff valuesintertumoral andintratumoral heterogeneity and test platform uniformitieshave limited their clinical applications therefore future strategies that combine different predictive featuresmay be more effective biomarkers for the accurate prediction of cancer immunotherapy response but need tobe carefully integratedsin summary we have successfully constructed a noveltmb estimation model using only genes that can beused to estimate the westmb and stratify survivalprognosis after radical surgery and clinical outcomes ofici therapy in nsclc patients thus a customized panelfor the targeted sequencing of these selected genes instead of wholeexome sequencing can be designed or utilized as complementary genes included in the currentngs panels consequently by using our model the costeffectiveness may be considerably improved makingrealization of cancer immunotherapy response more accessible in standard clinical settings 0ctian bmc medicine page of supplementary informationsupplementary information accompanies this paper at httpsdoi101186s12916020016948competing interestsno potential conflicts of interest were disclosed by the authorsadditional file table s1 data sets used to calculate westmb forthe study cohorts table s2 characteristics of the patients included inthis study table s3 candidate genes and related information tables4 genes and the corresponding cds length fig s1 flowchart ofthe construction of estimation model fig s2 the correlation of westmb and tmb as estimated by different gene panels fig s3 forestplots of hrs for os and dfs in the tcga and earlystage nsclcpatients study cohort fig s4 the performance of 23gene based tmbestimation model for the luad and lusc subtypes of nsclc tcgadata fig s5 forest plots of hrs for dfs in the earlystage nsclcpatients study cohort fig s6 forest plots of hrs for pfs of the nsclc patients in zs immunotherapy cohort fig s7 westmb is shownbased on muc16 a ttn b and pole c mutation status fig s8 thecorrelation of muc16 mutation status with overall survival a anddiseasefree survival b based on the earlystage nsclc patients figs9 the correlation of muc16 ttn and pold1 mutation status withprogressionfree survival pfs based on the rizvi cohort and ourimmunotherapy cohort fig s10 comparison of predictive performanceof response to icis by our 23gene panel with lyu™s 24gene paneladditional file the correlation of estimation models with genenumber to with os and dfs in the training setacknowledgementswe thank all patients that were involved in this study we also thankguoqiang wang jing zhao and shangli cai the medical department 3dmedicines inc shanghai people™s republic of china for their contribution tothe st

" heat shock transcription factor1 hsf1 was overexpressed to promote glutaminolysis and activatemtor in colorectal cancer crc here we investigated the mechanism for cancerspecific overexpression of hsf1methods hsf1 expression was analyzed by chromatin immunoprecipitation qrtpcr immunohistochemistrystaining and immunoblotting hsf1 translation was explored by polysome profiling and nascent protein analysisbiotin pulldown and m6a rna immunoprecipitation were applied to investigate rnarna interaction and m6amodification the relevance of hsf1 to crc was analyzed in apcmin and apcmin hsf1ˆ’miceresults hsf1 expression and activity were reduced after the inhibition of wntcatenin signaling by pyrvinium orcatenin knockdown but elevated upon its activation by lithium chloride licl or catenin overexpression thereare much less upregulated genes in hsf1ko mef treated with licl when compared with licltreated wt mefhsf1 protein expression was positively correlated with catenin expression in cell lines and primary tissues aftercatenin depletion hsf1 mrna translation was impaired accompanied by the reduction of its m6a modificationand the upregulation of mir4553p which can interact with ²utr of hsf1 mrna to repress its translationinterestingly inhibition of mir4553p rescued catenin depletioninduced reduction of hsf1 m6a modificationand mettl3 interaction both the size and number of tumors were significantly reduced in apcmin mice whenhsf1 was genetically knockedout or chemically inhibiteds catenin suppresses mir4553p generation to stimulate m6a modification and subsequenttranslation of hsf1 mrna hsf1 is important for catenin to promote crc development targeting hsf1 could bea potential strategy for the intervention of catenindriven cancerskeywords colorectal cancer catenin hsf1 translation mir4553p m6a rna modification correspondence wangx118zjueducn jinhczjueducn1department of medical oncology cancer institute of zhejiang university sirrun run shaw hospital school of medicine zhejiang university hangzhouchina2labortary of cancer biology key lab of biotherapy in zhejiang sir run runshaw hospital school of medicine zhejiang university hangzhou chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csong molecular cancer page of introductioncolorectal cancer crc is the third most common cancer with high mortality rate globally the accumulation of various genetic and epigenetic changes activatesmultiple oncogenic signaling critical for the pathogenesisof crc such as wntcatenin signaling pathway its activation will eventually initiate a transcriptiondependent oncogenic process to promote cell cycle progression and apoptosis resistance while the mechanismfor activated wntcatenin signaling to promote crcdevelopment has been wellexplored no therapeuticstargeting this pathway has been successfully developedin addition to proliferation activation and apoptosisresistance metabolism reprogramming is one of important hallmarks of cancer cells for example cancercells favor glycolysis instead of oxidative phosphorylationfor glucose metabolism even in aerobic conditionswhich was wellknown as warburg effect [ ] pyruvate the last product of glycolysis is converted into lactate rather than acetylcoa acetyl coenzyme a fortca tricarboxylic acid cycle or the krebs cycletherefore targeting enhanced glycolysis has been proposed as novel options in the prevention and treatmentof human cancers including crc however as a metabolism hub tca cycle is important in both energyproduction and biosynthesis therefore it needs to bereplenished by anaplerotic reactions such as glutaminolysis previously we reported that heat shock transcription factor hsf1 stimulated glutaminolysis toactivate mtor and promote crc development by upregulating the expression of glutaminase gls1 thecritical enzyme in glutaminolysis hsf1 expressionwas increased in crc and had a positive correlationwith shorter diseasefree survival dfs however theupstream mechanism for hsf1 overexpression in crcwas still uncleargene expression can be controlled by multiple processesincluding transcription mrna degradation translationand protein degradation while gene translation and protein degradation have been extensively investigated moreand more studies focused on mrna translation by exploring the effect of noncoding rnas such as micrornasmirnas and new modifications of mrna including n6methyladenine m6a modification [ ] mirnas canform a mirnainduced silencing complex mirisc toposttranscriptionally regulate gene expression by inhibiting capdependent initiation and stimulating mrna deadenylation [ ] on the other hand as one of the mostabundant modifications in mrna m6a modification ofmrnas usually promotes translation by recruiting initiation factors such as eif3 to the ² end of the mrna while mirnas and mrna m6a modifications play a distinct role in mrna translation the interplays betweenthem were not clarifiedin this study we found that activated wntcateninsignaling stimulated hsf1 translation to promote crcdevelopment byrepressing hsf1 mrnatargetingmir4553p to increase m6a modification of hsf1mrna therefore targeting hsf1 translation could be anew strategy for the intervention of crc and other cancers driven by activated wntcatenin signalingmaterials and methodscell antibodies and chemicalshuman crc celllines sw480 sw620 dld1 rkowere obtained from the american type culture collection atcc all cells were routinely cultured in rpmi invitrogen “ or dmem invitrogen“ supplemented with fetal bovine serumall cells were incubated at °c with co2 and humidity the following antibodies were used for western blotting and ihc hsf1 12972s cell signalingtechnology cst ab52757 abcam catenin 8480scst actin l cst flag f1804“ sigmacyclind1 ab134175 abcam cleaved parp1 9541scst mettl3 a8370 abclonal gls1 ap8809babgent pyrvinium p0027 licl cycloheximide r750107 chloroquine c6628 mg132 and pd150606 d5946 were purchased from sigmaaldrichinterfering rna sirnasirna mirna mimicsinhibitors transfectiontargeting cateninsmallmettl3 and micrornas were synthesized by genepharma shanghai china and ribobio guangzhouchina the sequence of these sirnas and mirnaswere listed in additional file table s1 sirnas andmirna mimicsinhibitors were transfected into cellsseeded overnight by lipo2000 invitrogen usa or lipofectamine rnaimax transfection reagentinvitrogenusa according to the manufacturer™s instructionsluciferase activity assaythe plasmid of catenin reporter was gifted from profximei wu zhejiang university for hsf1 activity assaya fragment containing x hse were synthesized andinserted into the pgl3basic vectors promega corporation usa the plasmid was cotransfected with prlrenilla and catenin sirna by using lipo2000 invitrogen usa or treatment with pyrvinium by xtremegene hp dna transfection reagent roche usa ²utr segment of the hsf1 was cloned by pcr andinserted into the vector pmirreporter promegathe mutation of mir455 binding sites in hsf1 ²utrwas generated by quick sitedirected mutagenesis“ stratagene usa the resultant plasmidswere cotransfected with prl renilla and mir455 mimicsby usinglipo2000 invitrogen usa h post 0csong molecular cancer page of transfection the luciferase activity was measured by thedualglo luciferase assay system promega corporation usachromatin immunoprecipitation chipchip analysis was conducted with the simplechip„¢ enzymatic chromatin ip kit cst usa antibodies usedwere antihsf1 12972s cst tcf7l2c48h11 cstand flag f1804“ sigma the primers used for thepcr analysis of precipitated dna were shown inadditional file table s2 for flagchip assay theflagcatenin vector was transiently overexpressed bytransfection after h the enrichment of flagcateninon col27a1 promoter was measured by the chip kitimmunoblotting and immunohistochemistryimmunoblotting and immunohistochemistry ihc assays were performed as previously reported for immunoblotting total proteins were extracted with ripabuffer supplemented with protease inhibitors rocheusa after heating the protein sample to “ °c for min celllysates were transferred to polyvinylidenefluoride pvdf membranes after the membranes wereblocked in milk primary antibody with gentle agitation overnight at °cfor ihc assay was performed in a tissue array containing cases of colonic tissues primary antibodies usedwere listed above the degree of immunostaining wasassessed by independent pathologists and evaluated byassigning a score of “ scores were defined as follows no staining faint staining moderate staining and strong staining final scores of and were regardedas low expression whereas scores of and consideredas high expressionapoptosis detectioncell apoptosis was measured by flow cytometry analysisand western blotting for flow cytometry analysis ofapoptosis cells were harvested and resuspended in μl x binding buffer μl fluorescein isothiocyanatefitc annexin v and propidium iodide pi bd biosciences usa were added to the cell suspensionand then incubated for min at room temperatureafter that the samples were attenuated with 400ul xbinding buffer and analyzed by acs caliburflowcytometerpuromycinlabellingto detect the change of nascent hsf1 synthesis x cells were plated in cm dish afterthe giventreatment cells were incubated with biotindcpuromycin nu925bios jena bioscience for hcells were lysed with np40 buffer mm trishclph mm nacl np40 glycerolcontaining 1x protease inhibitor cocktail after adequatecentrifugation the supernatant was incubated with 80ulstreptavidin sepharose beads ge17“ sigma byrotating at °c for h to overnight the mixture waswashed by np40 buffer for times and subjected towestern blotting using hsf1 antibodyseparatestranslatingpolysome profilingpolysome profilingor nontranslating mrnas on a sucrose gradient according tothe number of bound ribosomes as previously described in brief cells were grown to confluence before collection cells were incubated with μgml ofcycloheximide for min then cells are lysed by polysome buffer [ mmoll kcl mmoll mgcl2 triton x100 μgml cycloheximide mmoll heparin and uml rnase inhibitor takara 1x cocktail] for min on ice lysates were centrifuged rpm for min and the supernatant was layered onto a to sucrose gradient gradients were then centrifuged at rpm for min at °c and polysomebound fractions were collected using an isco densitygradient fractionation system isco lincoln ne withcontinuous monitoring based on a260nm wavelengththe rna in each fraction was extracted using trizol reagent invitrogen and analyzed by realtime rtpcrbiotin pull down assaybiotin pull down assay was performed as described previously cells were transfected with biotinylatedmir4553p probes for h and resuspended using lysisbuffer mm tris ph mm nacl mmmgcl2 uml superasein mm dtt igepal protease inhibitors lysates were incubated withprepared streptavidin beadsge healthcare yeasttrna sigma was used for blocking lysates at °c for h then washed times with binding and wash buffer mm trishcl ph mm edta m naclfinally the bound rnas were extracted and purified forqpcrrna immunoprecipitation rip assayrip assay was performed by magna riptm rnabinding protein immunoprecipitation kitmilliporeno17“ briefly × cells were lysed in μlrip lysis buffer and immunoprecipitated with antibodiesofinterest and protein g magnetic beads at °covernight followed by six times of washes in washingbuffer and protein digestion at °c total rna wasisolated and subjected to rtpcr analysis followingantibodies were used for rip n6methyladenosine synaptic mettl3 a8370 abclonal igg millipore 0csong molecular cancer page of rnasequencing2x106 wt mef and hsf1 ko mef were plated andcultured overnight following day cells were treatedwith mm licl for h cells were collected with trizol reagent the total rna was processed by nebnext®polya mrna magnetic isolation module to enrichmrna and the product rna was used for constructionlibrary via kapa stranded rnaseq library prep kitillumina sequencing libraries denatured by mnaoh to generate singlestranded dna as amplified insitu illumina cbot truseq sr cluster kit v3cboths gd4013001 illumina the ends of the generatedfragments were used to run cycles by the illuminahiseq sequencer all the experimental steps afterthe rna extraction were conducted in kangcheng biotechnology co ltd aksomics shanghai china rnasequencing was performed three timesanimal experimentsanimal care and experiments were conducted in compliance with institutional animal care and use committeeand nih guidelines the c57bl6 j mice and apcminmice were purchased from model animal research center of nanjing university marc nanjing chinahsf1 ko mice reported previously were used to generate apcmin mice hsf1ˆ’ subsequently groupsof mice wild type apcmin apcmin hsf1ˆ’ and apcmin treated with knk437 as previously reported were fed with highfat diet kcal fat for monthsthe intestine was dissected flushed with pbs and cutopen longitudinally along the main axis the number oftumors was counted and the sizes oftumors weremeasuredstatisticsall data were expressed as mean ± sd unless specifiedthe student™s ttest was performed for statistical significance analysis p value was considered as statistically significantresultscatenin activates hsf1 in crcin an effort to explore potential regulations of hsf1 wescreened chemicals generating a gene expression patternsimilar to hsf1 depletion by connective map httpportalsbroadinstitutecmapinterestingly a recently reported inhibitor of wntcateninsignaling pyrvinium had a similar effect on genomewide gene expression as hsf1 depletion fig 1aand additional file figs1ab moreover the expression signature related to wntcatenin signaling was positively correlated with the hsf1 signature fig 1b indicating a potential connection of hsf1 withwntcatenin signaling indeed pyrvinium attenuated[ ]the activity of a luciferase reporter driven by hsf1 binding sites hse heat shock response elements [ ]fig 1c and reduced the expression of wellknown transcriptional targets of hsf1 such as hsp90aa1 hspa4hspb1 and hsph1 fig 1d and additionalfile figs1c chromatin immunoprecipitation chip assayfurther confirmed the reduced interaction of hsf1 withits transcriptional targets fig 1e and additional file figs1d in consistence with pyrvinium knockdown ofcatenin by sirna also decreased hsf1 activity fig1f reduced the expression of hsf1 targets fig 1g andadditional file figs1e and attenuated the interactionof hsf1 with its targets fig 1i and additional file figs1f furthermore hsf1 targets were upregulated bythe potent gsk3 inhibitor licl in colorectal cancer cellline rko which had a low level of catenin expressionfig 1hto explore the biological relevance of hsf1 activationto catenin signaling we profiled gene expression ofwild type mouse embryonic fibroblasts wt mef andhsf1 knockout mef hsf1 ko mef before and afterlicl treatment ncbi geo gse151119 while only genes were upregulated in licl treatedhsf1 komef there were genes significantly upregulated inwt mef after licl treatment fig 1j among them genes displayed a dependence on hsf1 since theirexpression levels failed to be upregulated by licl treatment once hsf1 was depleted fig 1k in fact their expression levels had a high correlation with theexpression of a previously reported hsf1 signature fig1l furthermore genes had a hse heatshock response element hse within their promoter regions fig 1m and additional file meaning that theyare most likely bona fide targets of hsf1 indeed someof them such as tma16 dedd2 hspa9 and kif21a havebeen confirmed as the target of hsf1 by chipseqncbi geo gse57398 fig 1n and additional file figs1g taken together these results indicated that catenin can positively regulate hsf1catenin stimulates hsf1 protein translationto delineate how catenin regulates hsf1 we quantitated protein levels of hsf1 before and after inhibitingcatenin both pyrvinium and catenin depletion reduced the protein level of hsf1 fig 2a and b in contrast overexpression of exogenous catenin increasedhsf1 protein level fig 2c furthermore hsf1 proteinlevel was increased after activating wntcatenin signaling by licl treatment in both rko and mef cellsfig 2d in addition hsf1 expression correlates wellwith catenin expression in primary tissues fig 2e andf p chisquare test all of these data indicatedthat catenin upregulates hsf1 protein expression 0csong molecular cancer page of fig wntcatenin signaling activates hsf1 a chemicals influencing gene expression in a similar manner to hsf1 inhibition were screened byconnective map analysis b the correlation of wntcatenin signaling signature and hsf1 signature was detected by gepia c the effect ofpyrvinium on hsedriven promoter activity was explored by luciferase reporter assay d the effects of pyrvinium on the targets of hsf1 wereanalyzed by rtpcr e binding of hsf1 to the promoters of hsf1 targets in crc cells treated with or without pyrvinium was determined by chipf the luciferase assays of hse before and after catenin knockdown were shown as in c g and h the mrna levels of hsf1 targets with catenin knockdown or licl treatment were analyzed by rtpcr i binding of hsf1 to its targets promoter in crc cells before and after cateninknockdown was analyzed by chip j volcano plot displays differentially regulated genes in dhsf1 compared to wt parental cells with licl reddots indicate significantly regulated genes based on adjusted pvalue and logfold change logfc p log2fc k differential geneexpression analysis in wt and hsf1ˆ’ mef treated with licl were performed by rnaseq numbers of upregulated genes in two cells wereshown in venn graph l the correlation of putative hsf1dependent genes from k with reported hsf1 signature was detected by gepia mnumbers of putative hsf1dependent genes with or without hse in their promoters were summarized n representative hsf1 chipseqtracks ncbi geo gse57398 for hsecontaining genes are shown asterisks indicate p 0csong molecular cancer page of fig catenin stimulates hsf1 protein translation a the effect of pyrvinium on the protein expression of hsf1 was explored by westernblotting b the effect of catenin knockdown on hsf1 protein level was analyzed by western blotting c the protein level of hsf1 before andafter catenin overexpression was analyzed by western blotting d the effect of licl on hsf1 protein level in rko and mef was analyzed bywestern blotting e the expression of catenin and hsf1 in colorectal tissue was analyzed by immunohistochemistry staining f the correlationbetween catenin expression and hsf1 expression in colorectal tissue was analyzed by chisquare test p g the effect of catenindepletion on hsf1 with puromycin labeling was determined by western blotting h amount of hsf1 mrna in various polysome fractions wasanalyzed by rtpcrp however there were no apparent alterations in hsf1mrna level after catenin knockdown or pyrviniumtreatment additional file figs2a and s2b meanwhile the halflife of hsf1 protein was also not changedbefore and after catenin knockdown additional file figs2c inhibitors of proteasome autophagy and calpains all failed to reverse hsf1 protein downregulationinduced by catenin knockdown additionalfile 0csong molecular cancer page of figs2d all of these results implied that catenin affects hsf1 protein expression mostlikely via translationregulation therefore puromycin labeling assay wasemployed to monitor the synthesis of nascent hsf1 protein as expected the puromycin labeling of hsf1was reduced by catenin depletion fig 2g to furtherconfirm it monopolysome fractions from cytoplasmicextracts of crc cells before and after catenin depletion were collected by sucrose gradient centrifugationthe subsequent rtpcr analysis revealed that catenindepletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 2h in summary catenin upregulates hsf1 expression by stimulatinghsf1 protein translationhsf1 protein translation is regulated by mir4553pas micrornas mirnas play an important role inregulating the efficiency of protein translation we wondered whether hsf1 protein translation was regulatedby micrornas based on bioinformatics screening bytargetscan mirdb and starbase some micrornas including mir4553p mir2145p mir4315p mir184mir4903p and mir375 were proposed to target ²utr of hsf1 mrna fig 3a after functional validation by western blotting mir4553p and mir2145pbut not other micrornas were capable to suppress theexpression of hsf1 protein in crc cells fig 3b andadditional file figs3a however mir2145p but notmir4553p also reduced hsf1 mrna level additionalfile figs3b what™s more an inhibitor of mir4553prather than mir2145p rescued the downregulation ofhsf1 protein by catenin knockdown fig 3c and additional file figs3c indicating that mir4553p mightbe relevant to catenininvolved regulation of hsf1protein translation indeed mir4553p inhibited the activity of luciferase driven by wild type hsf1 mrna ²utr but not its mutant unable to bind mir4553p fig3d the interaction of mir4553p with hsf1 mrnawas further confirmed by biotin pull down assay fig3e based on the analysis of tcga data httpmirtvibmssinicaedutwthe expression of mir4553p islower in colon adenocarcinoma than in normal tissuesadditional file figs3d similarly qpcr analysis revealed lower levels of mir4553p in crc tissues than inadjacent nontumor tissues fig 3f additionally wehad confirmed high expression of hsf1 in the same cohort of human crc tissues previously indeedmir4553p expression was negatively correlated with theexpression of hsf1 fig 3g on the other handmir4553p similar to hsf1 inhibition as we reportedrecently reduced the expression of hsf1 targets induced the viability inhibition and apoptosis activation ofcolorectal cancer cells fig 3hj and additional file figs3eg the seed sequence of micrornas was important for targeting mrna by basepairing indeed the seed sequence mutant of mir4553p could notdownregulate the protein level of hsf1 additional file figs4a confirming the importance of mir4553p totarget hsf1 protein expression in a word mir4553ptargets hsf1 mrna ²utr to inhibit its translationm6a modification of hsf1 mrna stimulates its proteintranslationin addition to microrna mrna modifications such asn6methyladenosine m6a play important roles in theregulation of hsf1 translation interestingly we noticedthat the matching sites of mir4553p seed sequence inhsf1 mrna ²utr contains a typical motif of m6amodification fig 4a which was supported by bioinformatic analysis httpwwwcuilabcnsramp fig 4b andadditional file figs4b moreover we have done themerip sequencing in sw620 and found that the ²utr region of hsf1 has one m6a modification site intriguingly this sequence is completely complementary tothe seed sequence of mir4553p additionalfile figs4c pcr analysis after merip m6a rna immunoprecipitation further confirmed m6a modification ofhsf1 mrna fig 4c what™s more the activity of luciferase driven by the mutant hsf1 mrna ²utr whichwas unable to bind mir4553p but retains the m6amodification site sequence drach d a g or u r a or g h a u or c [“] was higher than theactivity of luciferase driven by wild type hsf1 mrna²utr fig 3dindicating the importance of m6amodification to hsf1 expression as the main component of the methyltransferase œwriter complex [ ]mettl3 was also bound to hsf1 mrna fig 4donce its expression was depleted m6a modification ofhsf1 mrna was decreased fig 4e in consistencewith its potential roles in promoting protein translationsuch a reduction of hsf1 mrna m6a modification reduced hsf1 protein expression fig 4f and nascenthsf1 protein synthesis fig 4g furthermore mettl3depletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 4h while hsf1mrna or the stability of hsf1 protein were not changed additionalfile figs4d and s4e moreoverhsf1 protein was decreased with the knockdown ofythdf1 which was the reader protein of hsf1 m6amodification fig 4i to sum up m6a modification ofhsf1 mrna was relevant to stimulate its translationcatenin suppresses mir4553p to increase hsf1 mrnam6a modificationnext we explored the interplay between mir4553p andm6a modification of hsf1 mrna both hsf1 mrna 0csong molecular cancer page of fig hsf1 protein translation is regulated by mir4553p a overlap of hsf1targeting micrornas predicted by targetscan mirdb and starbaseb the effect of mir4553p on hsf1 protein was analyzed by western blotting c the effect of mir4553p inhibitor on catenin knockdowninduced hsf1 downregulation was determined by western blotting d luciferase activity assay was used to analyze the effect of mir4553p onthe activity of ²utr with or without mir4553p binding sites p e the binding between biotinmir4553p and hsf1 mrna wasdetermined by biotin pull down assay p f expression of mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed by qpcr g the correlation of hsf1 protein and mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed h the effect of mir4553p on viability of crc cells was explored by mts assay i the effect of mir4553p on apoptosis of crc cells wasanalyzed using flow cytometry after pi and annexin vfitc double staining j apoptosis of crc cells treated with or without mir4553p wasdetermined by western blottingm6a modification and its binding to mettl3 were decreased by the overexpression of wild type mir4553pbut not its mutant unable to bind to hsf1 mrna ²utr figfigs5aand additional5afileinterestingly mettl3 depletion not only reduced m6amodification of hsf1 mrna fig 4e but also enhanced the interaction of mir4553p with hsf1 mrnafig 5b and additionalfile figs5b while the 0csong molecular cancer page of fig m6a modification of hsf1 mrna stimulates its protein translation a the sites of hsf1 ²utr binding to the seed sequence of mir4553pwas consistent with m6a rna modification elements œdrach b bioinformatic prediction of m6a modification in ²utr of hsf1 mrna c m6amodification of hsf1 mrna was analyzed by merip p d binding of mettl3 to hsf1 mrna was detected by rip p e m6amodification of hsf1 mrna with or without mettl3 depletion was analyzed by merip p f the protein level of hsf1 before and aftermettl3 depletion was detected by western blotting g the effect of mettl3 knockdown on hsf1 synthesis was determined by western blottingafter puromycin labeling h amount of hsf1 mrna in various polysome fractions was analyzed by rtpcrp i the effect of ythdf1knockdown on hsf1 protein level was analyzed by western blottingexpression of mature and primary mir4553p was notupregulated additional file figs5cd these resultsindicated that mir4553p may compete with mettl3for the m6a modification of hsf1 mrna thus inhibitinghsf1 protein translation furthermore the binding ofmir4553p to hsf1 mrna was not changed by ythdf1deletion additional file figs5e indicating that thetranslation repression of hsf1 mrna was more likely tobe mediated directly by the reduced m6a modification ofhsf1 0csong molecular cancer page of fig catenin suppresses mir4553p to increase hsf1 mrna m6a modification a m6a modification and mettl3 interaction of hsf1 mrnawith wt or mutant of mir4553p were analyzed by rip b the interaction between biotinmir4553p and hsf1 mrna with or without mettl3depletion was analyzed by biotin pull down c the effect of mir4553p inhibitor andor catenin knockdown on m6a modification of hsf1mrna was analyzed by merip d the effect of mir4553p inhibitor andor catenin knockdown on the interaction of mettl3 with hsf1 mrnawas analyzed by rip e the effect of catenin knockdown on interaction of mir4553p and hsf1 mrna was analyzed by biotin pull down f andg the levels of mature f and primary g mir4553p with catenin knockdown or licl treatment were determined by rtpcr h the correlationof col27a1 and mir455 was analyzed in linkedomics httplinkedomics i the effect of catenin depletion or licl on mrna level ofcol27a1 was analyzed by rtpcr j the interaction of catenintcf7l2 and hsf1 promoter was determined by chip k the correlation of catenin protein expression with the rna level of col27a1 was detected by linkedomics httplinkedomicsindeed mir4553pcatenindepletioninduced decrease of hsf1 mrna m6a modification fig 5c and additional file figs5f meanwhilethe interaction of mettl3 with hsf1 mrna wasinhibitorrescuedabrogated by depleting catenin fig 5d and additionalfile figs5g accompanied by the increased interactionof mir4553p to hsf1 mrna fig 5e and upregulationof mature fig 5f and additionalfile figs5h 0csong molecular cancer page of precursor additional file figs5i and primary mir4553p fig 5g and additional file figs5j in contrastwhen catenin was upregulated by overexpression or licltreatment both mature mir4553p fig 5f and additionalfile figs5h and primary mir4553p fig 5g and additional file figs5j were downregulatedprimary mir4553p was derived from a premirna hairpin encoded in intron of the collagen gene col27a1 additional file figs5k actually col27a1 expression was significantly correlated with the expression ofmir455 httplinkedomics fig 5h consistent withthis we observed col27a1 mrna levels were increasedupon catenin depletion while decreased after licl treatment fig 5i and additional file figs5l moreover catenintcf7l2 complex could interact with the promoterof col27a1 while the pair of primers negativechipprimer at a position far away from the promoter region couldnot enrich col27a1 fig 5j and additionalfile figs5m meanwhile the protein expression of cateninwas negatively correlated with rna level of col27a1httplinkedomics fig 5k these results indicatedthat the transcription of col27a1 was inhibited by wntcatenin signalingleading to decreased biogenesis ofmir4553p therefore catenin facilitates the shift frommir4553p binding to m6a modification in hsf1 mrnaby suppressing mir4553p expression eventually promoting hsf1 protein translationboth genetic and chemical inhibition of hsf1 attenuatecolorectal carcinogenesis in micein light of these in vitro findings we further exploredthe relevance of hsf1 to colorectal carcinogenesis inapcmin and apcmin hsf1ˆ’ mice since the interaction of mouse mir4553p and mouse hsf1 mrnaseems to be well conserved mouse mir4553p seed sequence cagucca the binding site in mouse hsf1mrna ²utr tggactg the expression of hsf1 andits downstream target gls1 were increased whilemir4553p expression was reduced in intestine tissuesfrom apcmin mice compared with normal c57bl6mice fig 6a and b after fed with highfat diet for months these apcmin mice developed multiple tumorsin the intestine fig 6c however both the size andnumber of tumors were significantly reduced in apcminmice treated with a chemical inhibitor of hsf1 knk437and apcmin hsf1ˆ’ mice fig 6d accompanied bythe downregulation of hsf1 targets fig 6e all of theseresults confirmed that hsf1 is a novel downstream target of wntcatenin signaling important to promotecrc developmentdiscussiongenetic changes in components of wntcatenin signaling such as deletion of the apc gene and ctnnb1mutations have been frequently detected in many typesof human cancers all of these muta

"The known molecular characteristics of each cell line are shown in . The 25 NSCLC cell lines consisted of 8 EGFR-mutant 6 KRAS-mutant 1 HER4-mutant 1 NRAS-mutant 1 PIK3CA-mutant 1 EML4-ALK fusion 1 HER2-amplified and 6 cell lines without gene alterations listed. Nine of the 17 EGFR-wild type cell lines were sensitive to Ad-REIC. HCC827 and its resistant subline HCC827-GR-high2 showed a similar degree of sensitivity to Ad-REIC. No trend in molecular genotype was seen between the sensitive and non-sensitive cell lines. These results suggested that the effect of Ad-REIC does not depend on a known molecular genotype. .0087900.g001 Sensitivity and predictive factors of sensitivity for Ad-REIC treatment in 25 NSCLC cell lines. The inhibition rates of 25 NSCLC cell lines transfected with Ad-REIC compared to Ad-LacZ are shown as black bar in 20 MOI and white bar in 200 MOI. Thirteen cell lines with over 40% inhibition rate in 20 MOI are defined as highly sensitive and 12 cell lines with lower inhibition rate in 20 MOI are defined as resistant. All the resistant cell lines shows over 40% inhibition rate in 200 MOI. The cell lines are classified into 3 categories based on the GRP and CAR protein expression level as follows; category A (low GRP/high CAR) category B (low GRP/low CAR or high GRP/high CAR) category C (high GRP/low CAR). All 8 highly sensitive cell lines were included in category A and all 5 resistant cell lines were included in category C. Sq; squamous cell carcinoma AD; adenocarcinoma LC; large cell carcinoma ADSQ; adenosquamous cell carcinoma MM; malignant mesothelioma NHF; normal human fibroblast. Hoechst 33342 staining was performed in A549 cells to examine the induction of apoptosis. Apoptotic cells were observed in Ad-REIC-treated A549 cells (a). The mean rate of apoptosis was 22% and it was significantly (p<0.001 by Cochran-Mantel-Haenszel test) increased in comparison with the control Ad-LacZ treatment. .0087900.g002 Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. (a) Induction of apoptosis after in vitro Ad-REIC treatment as examined in A549 cells using Hoechst 33342 staining. The upper panel indicates the appearance of apoptotic cells after Ad-REIC treatment. The lower panel shows the apoptotic rate of A549 cells after the indicated treatment. A total of 5 different fields were examined under a microscope to determine the apoptotic rate. A significant difference was observed (*p<0.001) between the Ad-LacZ and the Ad-REIC treatment. (bar: 100 µm) (b) Western blot analysis for proteins involved in signal transduction triggered by Ad-REIC. Cells were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20 MOI. (c) H460 cells which are resistant to adenovirus transduction were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20100 and 200 MOI. The effect of recombinant REIC/Dkk-3 protein on NSCLC cell lines was examined in 7 randomly selected cell lines (NCI-H522 NCI-H611 NCI-H1299 NCI-H1819 NCI-H2009 PC-9 and A549). The MTS assay showed that REIC/Dkk-3 protein did not affect cell viability in the examined cell lines when administered at a concentration ranging from 1 to 200 µg/mL (data not shown). Expression of GRP78 and CAR in response to Ad-REIC therapy As predictive factors of Ad-REIC sensitivity in NSCLC we examined the expressions of GRP78 and CAR; these expression statuses were correlated with the inhibition of cell viability by Ad-REIC in 13 cell lines. A previous study reported that the overexpression of GRP78 inhibited ER-stress which may be oppositely correlated with the effect of Ad-REIC. CAR expression is tightly associated with the efficacy of adenovirus infection which may be positively correlated with the effect of Ad-REIC. Western blotting was performed and the expression level was quantified as shown in and . The median (range) of GRP78 and CAR expressions were 0.24 (0.075“0.98) and 0.60 (0.080“2.1) respectively. Based on these data cells with a GRP78 expression level more than 0.25 were defined as High CRP78 expression while those with a GRP78 less than 0.24 were defined as Low GRP78 expression. Regarding the CAR 15 cell lines significantly high level of CAR expression (over 0.50) were defined as High CAR expression while 10 cell lines those with significantly low level of CAR expression (under 0.20) were defined as Low CAR expression. GRP78 expression was low in 8 of the 13 Ad-REIC-sensitive cells (62%) and in 4 of the 12 Ad-REIC-resistant cells (33%). CAR expression was high in 12 of the 13 Ad-REIC-sensitive cells (92%) and in 3 of the 12 Ad-REIC-resistant cells (25%). Next we classified the cell lines into three categories based on the GRP78 and CAR expression statuses; cells with a Low GRP78/High CAR expression were classified as Category A those with Low GRP78/Low CAR or High GRP78/High CAR expression were classified as Category B and those with High GRP78/Low CAR expression were classified as Category C. The high sensitive cell rates were 100% in Category A (8 out of 8 95% confidence interval [CI]: 63“100) 42% in Category B (5 out of 12 95% CI: 15“72) and 0% in Category C (0 out of 5 95% CI: 0“52) (). "

" in penicillin/streptomicine-free DMEM for 24 hs according to manufacturer's instructions. Protein extraction and Western Blotting Cell pellets were suspended in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Sigma Aldrich Corp. St Louis MO USA). The extracts were then clarified by centrifugation at 15000 rpm for 15 min at 4°C and the protein concentration was determined with the Bradford reagent (Bio-Rad Laboratories Hercules CA) and spectrophotometric analysis. Lysates were incubated in 5x sodium dodecyl sulfate (SDS) sample buffer (5 min 95°C). An amount of 10-30 µg of proteins for each sample was loaded onto 8“15% SDS polyacrylamide gel. Proteins were then transferred onto a nitrocellulose membrane. The membrane was blocked for 1h with non-fat dry milk in TBS containing 0.05% Tween 20 washed and successively incubated with different primary antibodies for 12h at 4°C. The membranes were then washed three times for 10 min and incubated with the HRP-conjugated secondary antibody for 1h at room temperature (RT). After a thorough washing the blot was exposed to ECL (GE Healthcare NJ) followed by autoradiography. The intensity of the bands was quantified using Image J software (NIH Bethesda MD). Sulphorhodamine (SRB) assay Cells were seeded in 96 well plates at a density of 3—103. The next day (day 0) one plate was assessed. The remaining plates were tested at 2-day intervals for a total of 6“8 days. Cells were fixed with 100 µL per well of ice-cold 40% (vol/vol) TCA (Sigma Aldrich Corp. St Louis MO USA) gently added on top of the medium overlaying the cells. The plates were then incubated for 60 min at 4°C. Wells were rinsed five times with tap water and then stained with 0.4% SRB solution (100 µl stain/well; Sigma Aldrich Corp. St Louis MO USA) for 30 min at RT. After staining SRB solution was removed unbound dye was removed by washing five times with 1% acetic acid solution and left to air dry. The bound SRB dye was then solubilized by adding unbuffered Tris-base solution (100 µl/well) and plates were placed on a plate shaker for 10 min at room temperature. Plates were then read at OD 492 nm using a microplate reader. 3D Overlay Culture on Matrigel Thawed Matrigel (BD Bioscience) in a volume of 70 µl/well was added into each of the wells of the eight-well glass slide chambers (Thermo Scientific) and spread to form a 1-mm thick bed. Matrigel was left to solidify at 37°C for 15 min. Then cells (1—103/well) were plated in medium containing 2% Matrigel and allowed to grow in a 5% CO2 humidified incubator at 37°C. Flow cytometry (FACS) After treatments 1—105 cells were collected washed in phosphate-buffered saline (PBS) pelleted by centrifugation and fixed in 70% ethanol. Immediately prior to staining the cells were washed twice in PBS and suspended in PBS containing 50 µg/ml of RNAse A (Qiagen S.p.A Milano Italy). The cells were stained with propidium iodide (final concentration 100 µg/ml) for at least 1 h at 4°C and analyzed using a LSR II flow cytometer (BD Biosciences). The percentage of cells in subG1 G0/G1 S and G2/M phases were determined from >10000 cells using the FACSDiva 6.0 software (BD Biosciences). Caspase - Glo® 3/7 assay Caspase-3/7 activation was measured using the Caspase-Glo 3/7 Luminescence Assay (Promega Corp. Madison Wisc. USA) according to the manufacturer™s instructions. In a 6 well plate 3—105 cells were incubated. The day after the cells were treated with siRNAs both with and without drugs for 48 h. Then the cells were collected by trypsinization and approximately 15—103 cells were transferred in a 96-well white plate. Caspase-3/7-Glo reagent was added and the samples were incubated at 37°C for 1 h. The luminescence that is proportional to the caspase 3/7 activities was determined by luminometer (Tecan Sunrise Austria GMBH). Transwell Cell Invasion Assay About 5—104 cells in 200 µl of ?-MEM (Gibco Life Technologies Monza Italy) were plated in the Matrigel-coated upper chambers of the 24-well Transwell invasion assay plate (Corning NY 14831 USA). Plates were incubated at 37°C for 48 h. Cells in the lower chamber (including those attached to the lower surface of the membrane) were fixed in 4% paraformaldehyde (VWR Milan Italy) stained with DAPI (Lonza Basel Switzerland) and counted with fluorescence microscope (Metamorph “ Axiovert microscope). Wound-Healing Assay About 25—103 cells were seeded in a 12-well plate and after 24 h transfected with siMSLN-1. A linear scratch in the confluent cell monolayer was made with a sterile pipette tip after 12 h (time optimized following preliminary trials) following siRNA transfections. Then cells were rinsed and incubated in full medium. Finally cells were inspected and fixed with paraformaldehyde after 36 h following the scratch. Cells were stained with crystal violet 0.1%solution (dissolved in 20% ethanol) to enhance contrast and photographed with a phase-contrast microscope at 10X magnification. The migration was then evaluated on the images and measured with Image J software. Statistical analyses The measurements of gene expression performed on cell lines and the results obtained from the in vitro assays were statistically evaluated using a two-tailed Student™s t-test. The effects of the combination of treatments (drugs +/“ siRNAs) were evaluated with a multifactor analysis of variance (MANOVA) model. The statistics were performed with the software R (http://www.r-project./) and Statgraphics Centurion XV (StatPoint Inc.). Results MSLN expression in MPM cell lines The expression level of MSLN was screened in Mero-14 IstMes2 and NCI-H28 human MPM cell lines. Met5A a non-malignant immortalized cell line was also screened and used as reference. The expression of MSLN mRNA in Mero-14 cells was higher than in Met5A (A). Western blotting supported these data showing high MSLN levels in Mero-14 cells (B) and low levels in Met5A. Similar to mRNA expression IstMes2 had lower levels of MSLN than Met5A.No levels of MSLN were observed for NCI-H28. Thus two different silencing-RNAs (siRNAs) were assayed: siMSLN-1 and siMSLN-2 for MSLN in Mero-14 cells (Table S2; C). For further experiments siMSLN-1 was employed given its better performance in gene silencing (>95% for siMSLN-1 D). .0085935.g001 Expression levels of MSLN in human MPM cell lines and Met5A. A. RT-qPCR showing the mRNA expression levels of MSLN measured on MPM cell lines and related to Met5A cells (set to 1). RPLP0 HPRT and TBP were used for normalization. Error bars show the standard error of the mean (SEM) from three independent experiments each performed in triplicate. Mero-14 cells showed the highest expression levels of MSLN (P?=?0.02). B. The protein levels of MSLN in Met5A Mero-14 IstMes2 and NCI-H28 cells. ?-actin was used as reference. The protein levels were confirmed by two independent experiments. MSLN is shown as a band at 40 kDa. C. RT-qPCR showing the endogenous mRNA expression levels of MSLN in Mero-14 cells related to their own siCtrl (set to 1). RPLP0 HPRT and TBP were used for normalization. Error bars are SEM from three independent experiments each performed in triplicate. The siRNA chosen for the analysis is: siMSLN-1 (40 nM; *P?=?0.002) active on Mero-14 cells. D. Protein levels of MSLN (shown as a band at 40 kDa) after depletion with siMSLN-1 and -2 (40 nM). ?-actin was used as reference. The protein levels were confirmed by three independent experiments. Role of MSLN in cellular growth The effect of MSLN silencing on cellular growth was evaluated by performing two different assays: the SRB assay and the 3D Matrigel-overlay model. The first facilitates the assessment of the number of cells grown in a bi-dimensional context at a given time [29] whilst the latter facilitates the evaluation of the dimension and shape of spheroids formed in a three-dimensional context. Following the administration of siMSLN-1 a significant reduction (p < 0.05) in the proliferation rate was observed for Mero-14 cells starting from the third day of treatment as compared to cells treated with control siRNA (siCtrl) reaching a 86% decrease at day 6 (A). This result was also corroborated by a reduced expression of the phosphorylated forms of AKT and ERK pAKT and pERK being markers of proliferation [30] (B). When MSLN was transiently over-expressed in the non-MSLN expressing NCI-H28 cells increased levels of pAKT and pERK were observed confirming a link between MSLN expression and proliferation (B). The silencing of MSLN in Mero-14 cells was also associated with smaller and uniform spheres (mean ?=? 34.4 µm±3.11) as compared to the cells treated with siCtrl (mean ?=? 52.5 µm ±7.65 p<10?6). About 72.5% of siCtrl-treated spheres and only 22.5% of siMSLN-1-treated spheres measured > 40 µm (C). Thus Mero-14 cells following MSLN silencing showed a low proliferation rate and a reduced capacity of forming spheroids. .0085935.g002 Role of MSLN in cellular growth. A. SRB proliferation assay in Mero-14 cells treated with 40 nM of the siCtrl or siMSLN-1 (*P ?=? <10?4). Error bars represent SEM of three independent experiments each performed in quadruplicate. B. Western blotting analysis of MSLN p-AKT p-ERK and ERK1-2 on Mero-14 cells treated with siCtrl or siMSLN-1 and on NCI-H28 cells transfected with an empty vector (pcDNA3.1) or a plasmid overexpressing MSLN (pcDNA3.1-MSLN). ?-actin was used as reference. The protein levels were confirmed with three independent experiments. C. The picture represents the phase contrast microscopy of Mero-14 cells cultured in 3D Matrigel-overlay chambers after silencing of MSLN (siMSLN-1 at 40 nM). Magnification 10X. Two different experiments were performed each in triplicate. The percentages of Mero-14 cells classified according to the dimensions of the spheres following treatments with siCtrl or siMSLN-1 in 3D Matrigel-overlay chambers were also reported. Legend to figure 2: Gray line?=? cells treated with siMSLN-1; Dark line?=? cells treated with siCtrl. Role of MSLN in cell cycle progression and apoptosis To examine the effects of MSLN silencing on cell cycle progression Mero-14 cells were treated with siCtrl and siMSLN-1 for 72 h and analyzed with flow cytometry. A statistically significant decreased share of Mero-14 cells in S+G2+M-phases was observed following MSLN silencing as compared to controls (Figure 3). The reduction (standardized for siCtrl) was approximately by 25%. Measured at 48 h after siRNA transfection the Mero-14 cell line did not show any changes in the activities of apoptosis markers: caspase-3 and -7. .0085935.g003 Figure 3 Progression of cells through the cell cycle following flow cytometry analysis. The graph shows the percentage of Mero-14 cells in phase S+G2+M treated with 40 nM of the siCtrl or siMSLN-1. The S+G2+M phase of the cell cycle was slightly reduced following the treatment with siMSLN-1 (*P?=? 0.033). Error bars represent SEM of six independent experiments. Role of MSLN in migration and invasion The effect of gene silencing on cellular migration was evaluated using the wound-healing assay (assessing the repopulation of a scratched area in a plate) [31].The invasiveness was measured using the trans-well assay (assessing the number of cells passing through the pores of the membrane) [32]. No statistically significant differences in migration parameters were observed in Mero-14 following the siRNA treatments (Figure 4A). However Mero-14 cells showed a statistically significant reduced invasion as compared to controls at 48 h after MSLN silencing (Figure 4B). .0085935.g004 Figure 4 Role of MSLN in cellular migration and invasion. A. No effects observed in the wound-healing assay following siRNA transfections. Confluent monolayers of Mero-14 cells transfected with 40 nM of siCtrl or siMSLN-1 respectively. Two different experiments were carried out each performed in triplicate. B. Trans-well cell invasion assay on Mero-14 cells transfected with 40 nM of the siCtrl (top) or siMSLN-1 (bottom). Pictures were taken using a fluorescence microscope at 10X magnification and are reported as negative of the originals to enhance the contrast between the background and the DAPI-stained cells. The bar chart shows the average of invasive cells (error bars represent SEM of two independent experiments each done in triplicate *P ?=? 0.0044). Role of MSLN in cellular growth following treatments with chemotherapeutic drugs After 6 days of treatment cisplatin used as a single agent caused a 26% reduction (not statistically significant) in the proliferation rate of Mero-14 cells. Then the effect of siMSLN-1 was evaluated in combination with cisplatin. When the two agents were used in combination the growth was completely inhibited (p<0.05 Figure 5A). Moreover the addition of siMSLN-1 in cultures treated with imatinib or gemcitabine (each as a single agent) or imatinib+gemcitabine caused further reductions in proliferation. However the effect of siMSLN-1 was not statistically significant in contrast to cultures treated with the two chemotherapeutic drugs together with siCtrl (p?=?0.21 p?=?0.38 and p?=?0.17 respectively). "

objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anti‘cancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often car‘ried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and prolif‘eration of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht‘ cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht‘ cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell context‘dependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0csainz‘cort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoy™s 5a medium alamarblue® ab invitrogen were bought leibovitz™s l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillin“streptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at ˆ’ a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoy™s 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research l™hospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillin“streptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at “ cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapiro“wilk test to assess data normality and nonparametrical independent samples kruskal“wallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht‘ cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were ‰ a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd ‰ a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainz‘cort a0et a0al bmc res notes page of fig a ht‘ cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht‘ cells incubated with or without μm cbd for h representative images are shown bar µm c ht‘ cell viability according to dapi staining see the œmaterials and methods section ht‘ cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapi‘positive cells dead cells showing a higher percentage in cbd‘treated cells a representative experiment c is shown p viability and a0proliferation of a0ht‘ cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainz‘cort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht‘ cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst‘ the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainz‘cort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd “ a0μgml reduced the viability of cancer cells and also had effects on other survival variables [“ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu ‘bromo‘²‘deoxyuridine cbd cannabidiol dapi ²‘diamidino‘‘phenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors™ contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag wald‘sch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltec‘ub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implica‘tions in cancer metabolism trends cancer “ https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtner‘feigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w mueller‘klieser w renner k kreutz m ldha‘associated lactic acid production blunts tumor 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sensors “ https doi103390s1209 scott ka dalgleish ag liu wm anticancer effects of phytocannabinoids used with chemotherapy in leukaemia cells can be improved by altering the sequence of their administration int j oncol “ śledziński p zeyland j słomski r nowak a the current state and future perspectives of cannabinoids in cancer biology cancer med “ https doi101002cam41312 solinas m massi p cinquina v valenti m bolognini d gariboldi m monti e rubino t parolaro d cannabidiol a non‘psychoactive cannabinoid compound inhibits proliferation and invasion in u87‘mg and t98g glioma cells through a multitarget effect plos one 2013810e76918 https doi101371journ alpone00769 sreevalsan s joseph s jutooru i chadalapaka g safe sh induction of apoptosis by cannabinoids in prostate and colon cancer cells is phos‘phatase dependent anticancer res “ tomko a o™leary l trask h achenbach jc hall sr goralski kb ellis ld dupr© dj antitumor activity of abnormal cannabidiol and its analog o‘ in taxol‘resistant preclinical models of breast cancer front phar‘macol https doi103389fphar van der valk j bieback k buta c cochrane b dirks wg fu j hickman jj hohensee c kolar r liebsch m pistollato f schulz m thieme d weber t wiest j winkler s gstraunthaler g fetal bovine serum fbs past”pre‘sent”future altex “ https doi1014573 altex wu h‘y huang c‘h lin y‘h wang c‘c jan t‘r cannabidiol induced apoptosis in human monocytes through mitochondrial permeabil‘ity transition pore‘mediated ros production free radical biol med “ https doi101016jfreer adbio med201806023publisher™s notespringer nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 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" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[“] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [“] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [“] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics„¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturer™s protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturer™s protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells — per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx„¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx„¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe ˆ’δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgaccˆ’ ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse ™tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells “ confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondary™s for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [“] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [“] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3™utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors™ contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res “aberle dr berg cd black wc the national lung screening trialoverview and study design radiology “ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lett“zhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab ““ wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab ““lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res “ wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res “qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med “ hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer “ 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget “ cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med “ zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract “ huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sci“liu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci “ yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep “sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res“ mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun “su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer res“liu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother “ yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother“ duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai“ yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai “li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin “li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a “ bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene “li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep “ qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagan™s nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming People™s Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expˆ’b·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expˆ’bD f · expˆ’bD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [“] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [“] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Begg™s test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deek™s plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Begg™s testor Deek™s test indicated the potential of publication bias[] Inconsistency index I2 and Cochran™s Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagan™s nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies“ scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The χ2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ““ “ ± “NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA “ ± NA “ ± “Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of ˆ’ mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Begg™s Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of Begg™sTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of ˆ’ P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Begg™s Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Begg™s Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deek™s funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagan™s nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks™ funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagan™s nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors™ contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors™ informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming People™sHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol “ httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med “ httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology “ httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol “ httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis “httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L

" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients™ travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [“] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patient™s condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as folˆ’ mmhglows ph paco2 mmhg hco3base excess be ˆ’ meql and lactate mmoll the patient™s laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patient™sgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patient™s left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [“] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patient™s lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patient™s leg the patient™s leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ‰¦s s s ‰¦s ‰¦s ‰¦s ‰¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patient™s vital signs during days “ ofhospitalization b changes in patient™s blood biochemistry during days “ of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors™ contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond “ hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr “ dobrović k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr “jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp “ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol “su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect “tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev “li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun “dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol “isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz “hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med “deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am “ wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med “ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis “ cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc “tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am “ changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg“ maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond “ ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol “ 0c"

complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen People™s Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s“1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [“] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[“] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [“] Following these discoveries The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions [“]The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [“] and a Cterminalregulatory tail [“] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [“] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ “] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ “] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [“] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins [“]It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [“ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [“] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions [“]its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [“] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [“] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [“] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies [“]Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as œundruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[“] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain œundruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ “] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ “] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after “months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [“] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [“] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [“] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [“] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [“] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPK™ssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPK™s regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [“] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[“]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [“] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ “] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous [“]YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucells™latorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic

" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since “ beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on people™s health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of today™sdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and [“]in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rrˆ’¾ ¾ pexp 02 rrˆ’°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso “ times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm [“]the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in “ sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in “disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungary™s gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the country™s gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of “ billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that “ of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary “ in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary “medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ “] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amountˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ total amountˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the population™s health status is responsible for about “ ofeconomic growth [“]in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases™ burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifa™s depression costs account foronly “ in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto people™s daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects “ such as the high costof sports injuries high rates of childhood illness “ haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since “ beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifa™s colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors™ contributionspa was the leader of the complete research coordinated the different coauthors™ work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev “reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med “ who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379“tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab “rishiraj n inactivity a bad œhabit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle “ gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev “ acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed “ hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©g“befektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health “katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj “ 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed “ andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c

the european commission asked efsa for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identified a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identified no reference point for chronic exposure could be identified from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufficient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientific output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientific outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientific opinion “ risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodifications or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientific opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when finalising the scientific opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic profiles of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspecific effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identified from these studies reduced postnatal survival of pups due to insufficientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufficient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identifiedin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufficiency cardiac failure coma and death have beenreported doses in the range of “ mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufficient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufficient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identifiedregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentifiedoccurrence data were only available for asolanine and achaconine and mostly for ˜maincroppotatoes™ and ˜new potatoes™ few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence of˜new potatoes™ with that of ˜maincrop potatoes™ and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the final processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby “ boiling in water and blanching of peeled potatoes by “ and frying in oil of peeledpotatoes by “ microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by “ and by “ respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the confidence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to “ of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identified uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows™ milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identified due to an insufficient database on the adverse effects of gas inruminants pigs poultry rabbits and fish an acute reference dose could not be derivedpotatoes are not grown specifically as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufficient to perform an exposure assessmentthus no risk characterisation could be performed due to insufficient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identified to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identified to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classification methodology for exposure assessment methodology for risk characterisation assessment hazard identification and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed influence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the influence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identification and characterisation summary of uncertainties conclusions hazard identification and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a “ major glycoalkaloids and their aglycones present in solanum species appendix b “ identification and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c “ details of the study design of the toxicokinetic studies appendix d “ comparison of developmental toxicity of single dose studies appendix e “ inhibition of cholinesterases by gas appendix f “ rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g “ studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h “ additional scenario for the human risk characterisation annex a “ occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are “ times greater in the peel than in the flesh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include flulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientific opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid profile ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of specific groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including specific groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no specification on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the first alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l‚¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well definedtheconfiguration is 22r 25stheitconfiguration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversification is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe prefix alpha a refers to the intact glycoside while the prefixes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s