During the COVID-19 pandemic, different SARS-CoV-2 variants of concern (VOC) with specific characteristics have emerged and spread worldwide. At the same time, clinicians routinely evaluate the results of certain blood tests upon patient admission as well as during hospitalization to assess disease severity and the overall patient status. In the present study, we searched for significant cell blood count and biomarker differences among patients affected with the Alpha, Delta and Omicron VOCs at admission. Data from 330 patients were retrieved regarding age, gender, VOC, cell blood count results , common biomarkers , ICU admission and death. Statistical analyses were performed using ANOVA, the Kruskal–Wallis test, two-way ANOVA, Chi-square, T-test, the Mann–Whitney test and logistic regression was performed where appropriate using SPSS v.28 and STATA 14. Age and VOC were significantly associated with hospitalization, whereas significant differences among VOC groups were found for WBC, PLT, Neut%, IL-6, creatinine, CRP, D-dimers and suPAR. Our analyses showed that throughout the current pandemic, not only the SARS-CoV-2 VOCs but also the laboratory parameters that are used to evaluate the patient’s status at admission are subject to changes. Among these categories, variants of interest (VOI) are those that present characteristics that warrant continuous monitoring and further evaluation, while variants of concern (VOC) are the variants that are proven to be more virulent, more transmissible, more pathogenic and at some extent may evade treatment options and/or the immune responses due to vaccination or previous infection , coagulative parameters , biochemical parameters  and inflammatory parameters . Other parameters, such as procalcitonin, KAccording to the dynamic nomenclature for SARS-CoV-2 lineages  and the The laboratory diagnosis of COVID-19 was performed on nasopharyngeal samples by real-time PCR, using either the NeuMoDxTM SARS-CoV-2 assay  or the Abbott Molecular Real-Time assay . The assay selection for each case depended upon the priority given during the everyday clinical practice for the respective sample. Testing of samples with higher priority was performed using the NeuMoDxTM SARS-CoV-2 assay.Viral RNA was extracted from nasopharyngeal samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation kit in the automated KingFisher Flex platform  according to the manufacturer’s protocol. A total of 10 μL of the extracted RNA was used for the library construction with the QIAseq FX DNA Library Core Kit and the QIAseq SARS-CoV-2 primer panel . Library quantification was performed with the KAPA SYBR FAST Universal qPCR kit , and the samples were paired-end sequenced on the NextSeq500 Illumina platform with an additional step of heat denaturation of libraries before loading. The resulting sequenced reads were 150 bp—long, and they were quality-controlled using the FastQC software v.0.11.9 .https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2 last accessed on 5 March 2023).Raw sequencing data were first processed using Trim galore for adapter and quality trimming. The cleaned reads were then mapped to the SARS-CoV-2 reference genome  using Minimap2 tool with a minimal chaining score equal to 40. From this process, only the paired-end sequences were retained, while any other  were removed. The produced alignment was processed  using the samtools/bedtools software tools suite. GATK Haplotype Caller  was used to detect and filter nucleotide mutations present in over 90%  of the sequences at each position of the reference genome. The detected mutations were used to create consensus sequences for each sample (bcf tools consensus), which were used as input to the Pangolin tool for phylogenetic identification and assignment of different SARS-CoV-2 strains. Ultimately, all identified mutations were annotated using the SnpEff tool and the NC_045512.2 (v.5.0) database  of EMBL-EBI (accession number PRJEB44141). Sample accession numbers are provided as ®, Brea, CA, USA). The suPAR was determined by the suPARnostic® TurbiLatex assay  on the Cobas® 8000 c702 analyzer  using K3-EDTA plasma samples. The measurement of IL-6 was performed on the Cobas® e 411  in serum samples. D-dimers were measured on the ACL TOP 750 CTS  coagulation analyzer in plasma samples retrieved from whole blood, drawn into buffered sodium citrate  tubes after centrifugation at 3000 rpm for 10 min.Blood cell analysis was performed on XN 1000 hematology analyzer , using whole blood samples in tubes with K3EDTA (Ethylene Diamine Tetra Acetic acid) as anticoagulant. Measurements of urea, creatinine, SGOT, SGPT and CRP were done in serum samples on the DxC 700 AU instrument  and the Kruskal–Wallis test  in order to examine the association of lab indexes and COVID-19 subtypes for the entire group of patients, and moreover, we performed the same analysis separate for male and female patients. Patients were categorized based on their age into three groups . Two-way ANOVA was performed to explore the interaction between age groups and COVID-19 subtypes on the values of lab indexes. Two-way ANOVA was also performed to examine possible associations between the age of the patients and the interactions between clinical outcomes and COVID-19 subtypes. Chi-square tests were performed to examine the association between COVID-19 subtypes and the clinical outcomes of interest . T-tests  were performed to evaluate the association between the age of the patients and the clinical outcomes. Finally, logistic regression models were performed to explore the effect of age on clinical outcomes, with and without adjusting for the different COVID-19 subtypes. All analyses were performed using IBM SPSS v.28 and STATA 14 .The study group included 330 patients  with a mean age of 55.2 years . Overallp-value = 0.020). More specifically, higher WBC values were found in Omicron cases (7.59) compared to Delta (6.12) . More specifically, Alpha showed a higher % of neutrophils (69.6) than Omicron . More specifically, statistically significant differences between Delta (0.61) and Alpha (1.07) were observed . Omicron presented higher PLT counts (247) than Alpha  and Delta  as well . Participants with the Delta subtype (5.92) had higher creatinine levels compared to participants with the Alpha (1.01) and the Omicron (1.67) subtypes . Participants with the Alpha subtype (48.3) had higher IL-6 levels compared to participants with the Omicron (31.9) subtype . The two-way ANOVA analysis proved that this difference occurred mainly in the patients of the Alpha subgroup. In the subgroup of Omicron, the Neut% was almost the same for patients >70 and patients 50–70 y. Similarly, in the Delta subgroup, the Neut% was almost the same for patients of 50–70 y and patients <50 y. Alpha patients also had higher Ig values (1.07) compared to Delta patients , and Delta patients presented higher creatinine values (5.92) than both Alpha  and Omicron  patients.The two-way ANOVA analysis proved that the Neut% difference was mainly due to the group of participants aged >70 y. In the group of participants aged 50–70 y, the difference of Neut% between Alpha and Omicron was almost zero. Additionally, in the younger age group (<50 y), the patients with Alpha had lower Neut% values compared to patients with Delta. We also observed that the main effect of age was statistically significantly associated with the Neut%  association between SARS-CoV-2 VOC and hospitalization was found. Indeed, 44.4% of the Omicron patients admitted to our hospital needed hospitalization, whereas 75% of Alpha and 70.6% of Delta patients were hospitalized. A significant association with ICU admission (p = 0.002) was also found. 30% of the patients with Alpha needed ICU admission, whereas only 16% of Omicron and 10% of Delta patients were admitted to the ICU. This similarity between Omicron and Delta highlights that even though Omicron cases were less likely to be hospitalized, severe Omicron cases could end up in the ICU with similar probabilities to Delta. No association was observed between VOC and death as a final outcome.A statistically significant . Delta-infected male patients had higher creatinine values (1.51) than Alpha patients . Among females, Omicron patients had higher WBC counts (7.71) than Delta  and lower IL-6 values  than Alpha  and Delta . CRP values were higher for female patients with the Delta (5.10) than for those with the Omicron  variant.Among male patients, those infected by Omicron had higher WBC (7.5) and PLT (233) counts than those infected by Delta  (p < 0.001)  A. As exp< 0.001) B. ParticNo statistically significant interactions between hospitalization and VOC on age were found. In other words, age was statistically significantly associated with hospitalization , but this association did not change for different SARS-CoV-2 VOCs. That means that despite the VOC, older patients are more likely to be hospitalized. No statistically significant interactions between ICU admission and VOC on age were detected. The same was observed for death and VOC on age.In the logistic regression analysis, however, age was statistically significantly associated with the hospitalization status of patients when adjusting for VOC. If the age was increased by one year, the risk of hospitalization increased by 6% for patients with the same VOC. Moreover, for patients of the same age, those with the Alpha VOC had a 4-fold higher risk of hospitalization compared to patients with Omicron, and those with Delta had a 3.8-fold higher risk of hospitalization compared to patients with Omicron. Age was significantly associated with death when adjusting for VOC. If the age was increased by one year, the risk of death increased by 8.3% for patients with the same VOC.p < 0.001). We observed that D-dimers differed significantly among the three VOCs . Patients with Omicron had higher mean values of D-dimers compared to patients with the other VOCs. Two-way ANOVA analysis proved that the main reason for this difference was due to the group of participants aged >70 y, where these differences were greater compared to the other age groups (p < 0.001). Two-way ANOVA analysis proved that the main reason for this difference was caused by patients with Omicron, where the differences between the three age groups were higher.Statistically significant interactions were found among VOC and age groups on D-dimers  were observed. The suPAR values differed significantly among the three VOCs (p < 0.001), and patients with Omicron had higher mean values of suPAR compared to the patients with the other VOCs. Two-way ANOVA analysis proved that the main reason for this difference was (due to) the group of participants aged >70 y, where these differences were greater compared to the other age groups (p < 0.001). Two-way ANOVA analysis proved that the main reason for this difference was the patients with Omicron, where the differences between the three age groups were greater, and also the Delta subgroup patients aged <50 y who had higher suPAR values compared to patients aged 50–70 y.Statistically significant interactions among VOC and age group on the suPAR  emerged and prevailed within the range of two weeks. Delta sub-variants, however, continued to be detected at lower rates. Omicron 2 (BA.2 and BA.2.9) emerged in February 2022 and became rapidly predominant within the next months .During the pandemic, it has been evident that infections with SARS-CoV-2 are associated with a wide range of symptoms. Moreover, a large study from England  showed cAt first glance, our results showed that together with the evolution of SARS-CoV-2, its interaction with the human organism is subject to changes, and this is reflected in variations regarding the values of different useful biomarkers for COVID-19. Some of our results seem to be confirmatory, like the fact that Omicron patients were less likely to be hospitalized than Alpha and Delta patients. Indeed, Alpha and Delta patients were four- and 3.8-fold more probable to be hospitalized than Omicron, respectively, and this underlines the value of molecular epidemiology surveillance to clinical practice and patient management. On the other hand, the elevated Omicron ICU admissions or the lack of significant association between VOCs and death should be considered, also taking into account that in our sample, the vaccination status and information on comorbidities were not available. The same limitations could also partially explain the fact that in our study, age was related to hospitalization and death but not to ICU admission.Interestingly, in our study group, Omicron was related to elevated PLT counts and D-dimer values in the elderly, suggesting that thromboembolic events may be more likely to occur in such patients. According to estimates, thrombosis accounts for one in every four fatalities around the world, making it one of the main causes of mortality in the globe . After 2SuPAR is the soluble form of uPAR, and elevated levels can be found in infections , neoplasmatic and autoimmune diseases. It has already been shown that high levels of suPAR are observed in COVID-19 patients and especially those with severe or critical illness, who had longer hospitalization and higher demands of oxygen therapy . SuPAR pFrom the early stages of the pandemic, the need to interpret the results of fast and easy-to-perform blood tests in the context of COVID-19 severity and/or outcome was evident. For this reason, several systematic reviews aimed to collectively assess the results of various individual research works. In a systematic review by Tjendra et al. , severalIn a later systematic review and meta-analysis , lymphopIn a systematic review and meta-analysis regarding the role of IL-6 in COVID-19, it was shown that higher levels of IL-6 were associated with poor clinical outcomes . More spAnother systematic review and meta-analysis investigated the role of biomarkers and attempted an estimation of threshold values that could be related to adverse clinical outcomes . HypoalbIn a systematic review and meta-analysis by Zhang et al. , the rolIn a study by Wang et al. , the corThe need for creating a risk tool based on the results of routine blood biomarkers and basic patient information that would ensure the best possible triage for COVID-19 patients led to the development of LUCAS, a COVID-19 mortality prediction calculator . LUCAS uOur study presents some limitations mainly because of its retrospective nature. Obviously, the outcome and the laboratory test results of COVID-19 patients may be influenced by viral coinfections and/or superimposed bacterial and fungal infections, especially during hospitalization . Even thDespite these limitations, to our knowledge, this is the first study to compare blood parameters among patient groups affected by different VOCs documented by whole genome sequencing data. These findings contribute to the growing literature on COVID-19 as different variants emerge. Overall, our results showed that each VOC combines with a unique “laboratory” identity. This fact may affect the utility of laboratory markers for monitoring COVID-19 activity and progression. In the same context, the therapeutic guidelines based on laboratory values could be adjusted for distinct VOCs. Altogether, perpetual updating of knowledge about the distinctive laboratory values of each VOC is vital for clinicians setting the personalized and targeted therapy of all COVID-19 patients as an ultimate goal.Our data, within the context and limitations of a real-world study, show that during the course of the pandemic, not only SARS-CoV-2 variants but also the laboratory parameters that are used to evaluate the patient’s status at admission may be subject to changes. The regular monitoring of VOIs and VOCs may provide essential information to clinicians about the circulating variants and their clinical importance. Medical doctors, however, should also be aware that the usefulness of some biomarker values may vary as variants evolve over time. Therefore, uninterrupted research and continuous medical education are needed to provide the best healthcare services to patients affected by COVID-19.


PURPOSE: To explore the relationship between ATM, ATR and CAT polymorphisms and prognosis of lung cancer patients received platinum-based chemotherapy.METHODS: 404 patients with lung cancer who received platinum-chemotherapy were enrolled and DNA typing was performed. Cox regression analysis and stratification analyses was performed to assess relationships between OS and PFS with SNPs genotypes. The prognosis of lung adenocarcinomaand squamous cell carcinomapatients was analyzed with The Cancer Genome Atlas (TCGA) database according to the grouping of CAT expression.RESULTS:CAT rs769217 was significantly related to PFS of patients with lung cancer who received platinum-chemotherapy. In the Additive model, rs769217 was associated with PFS . In the Dominant model, CT and TT genotypes led to lung cancer progression 0.738 times more than CC genotype. In stratification analyses of association between CAT rs769217 polymorphisms and PFS, the HR of patients at stage IV in additive model was 0.73, and HR was 0.745 (p = 0.034) in dominant model. For OS analyses, HR was 0.672 in the older lung cancer patients (>55 years old) in additive model. Meanwhile, in the Dominant model, it was found that the older patients with CT and TT genotypes had better prognosis, and the risk of death after receiving platinum-based chemotherapy was 0.692 times that of patients with CC genotype (p = 0.037). TCGA data shows that LUAD patients with high CAT expression have longer OS (p = 0.020).CONCLUSION:CAT rs769217 is significantly related to PSF of platinum-based chemotherapy in lung cancer patients and may be a biomarker for predicting the prognosis of lung cancer patients with platinum-based chemotherapy. ATM (ataxia telangiectasia mutant gene), ATR (ataxia telangiectasia and Rad3 related) and CAT  polymorphisms may be related to the prognosis of lung cancer patients receiving platinum-chemotherapy[Lung cancer is still the main cause of cancer death in the worldwide. There were an estimated 2,206,771 new cases and 1,796,144 cancer deaths of lung ancer worldwide in 2020 according to GLOBOCAN 2020. Platinumotherapy–10.via their downstream targets[ATM and ATR may be potential targets to affect platinum chemosensitivity[ATM and ATR SNPs may regulate kinase to enhance the DNA-damaging effect of Pt-based chemotherapy in cancer cells[ATM and ATR kinases were the key mediators of DNA damage response (DDR), which induce cell cycle arrest and facilitate DNA repair m targets–13. Obvinsitivity,15. Resensitivity. ATM andcer cells,16,17.2O2, reducing the production of ROS in cells[CAT rs769218 is related to the prognosis of gastric cancer patients receiving platin and fluorouracil-based adjuvant therapy, but the correlation between the prognosis of lung cancer patients and CAT polymorphism has not been reported[ATM, ATR and CAT with platinum-based chemotherapy outcome of lung cancer patients.Platinum-based chemotherapy causes cancer cells death through inducing oxidative stress to highly toxic level. Emergin in cells. Studies2) or carboplatin (AUC 5), which were both administered on Day 1 every 3 weeks, in combination with pemetrexed (500 mg/m2) on Day 1 every 3 weeks, gemcitabine  on Days 1 and 8 every 3 weeks, paclitaxel (175 mg/m2) on Day 1 every 3 weeks, docetaxel (75 mg/m2) on Day 1 every 3 weeks, or navelbine (25 mg/m2) on Days 1 and 8 every 3 weeks. Patients did not undergo surgery, targeted therapy, radiotherapy, or other anti-tumor therapy before chemotherapy. A physical examination as well as a detailed inquiry into each patient’s medical history was carried out. Patients with serious concomitant diseases that might greatly affect their physical condition were excluded. The study protocol was approved by the Ethics Committee of Institute of Clinical Pharmacology, Central South University , and all subjects were provided with written informed consents. We applied this study for clinical admission in the Chinese Clinical Trial Register (registration number: ChiCTR-RO-12002873)[All patients in this study were recruited from Xiangya Hospital of Central South University and Hunan Cancer Hospital , from 2012 to 2019. All patients were diagnosed with NSCLC by histopathological examination and confirmed the absence of driver genetic alterations that could be targeted. All patients received first-line platinum-based chemotherapy regimens for two to six cycles: cisplatin (75 mg/m12002873).http://pngu.mgh.harvard.edu/purcell/plink/)to detect and control the quality of SNPs data, requiring that the MAF  ≥0.05, the call rate≥90%, and conform to Hardy-Weinberg equilibrium. Finally, we selected 7 SNPs of ATR, ATM, and CAT for follow-up  and stored at −20°C according to the instructions. Each DNA sample was genotyped by Sequenom Mass Array Genotype Platform . After polymerase chain reaction (PCR), the product was purified by resin and analyzed by the Mass Array system (Sequenom). Then weollow-up .p-values were two-sided, p < 0.05 were supposed to be significant. All the above analyses were performed by The SPSS version 25.0 . UCSC Xena  was used to analyze the prognosis of LUAD and LUSC patients in TCGA database.OS  was defined as the time from treatment initiation until either the date of death, the date of last follow-up or the date of end of analysis. PFS  was defined as the time from treatment initiation until disease progression or death, whichever occurred first. Kaplan-A total of 404 lung cancer patients received platinum-based chemotherapy were included in this research. There were 175 patients with age ≤55 years old, accounting for 43.32%. Most of the patients were male, accounting for 77.48% (313). 40.10% (162) patients have the habit of smoking. 97.52% of the patients (394) were in stage III or IV. 50% of the patients (202) were adenocarcinoma and 42.33% (171) were small cell carcinoma .CAT rs769217 was significantly related to PFS. In the additive model, rs769217 was associated with PFS, HR = 0.747, 95% CI = 0.581–0.960, p = 0.023. In the dominant model, CT + TT genotypes led to lung cancer progression 0.738 times more than CC genotype. In the recessive model, the p-value > 0.05, which is not significant . In the Dominant model, the HR of patients in stage IV was 0.745, p = 0.034 (p = 0.031 of the older lung cancer patients (>55 years old) was found in the Additive model. Meanwhile, in the Dominant model, it was found that the older patients with CT and TT genotype had better prognosis, and the risk of death after receiving platinum-based chemotherapy was 0.692 times that of patients with CC genotype (p = 0.037)  .In 2019, about 2 million people worldwide died from lung cancer, more than any other cancer. Annuallin vitro studies lead to acute apoptosis that involves induction of oxidative stress but is largely DNA damage-independent[DNA is usually considered as the main target of platinum chemotherapeutic drugs, but the cisplatin used dependent. Celluladependent. Howeverdependent. ROS candependent.CAT rs769217 may affect the PFS of lung cancer patients receiving platinum-based chemotherapy , is a key antioxidant enzyme in the bodies defense against oxidative stress[CAT rs1001179 polymorphism and prostate cancer[CAT rs769217 can affect the prognosis of patients with biliary tr an act cancer (BTC), that knockdown of CAT induced chemoresistance through elevation of ROS level and activation of Nrf2-ABCG2 pathway in BTC cell lines[CAT polymorphism on the prognosis of patients with lung cancer receiving platinum chemotherapy has not been reported. CAT polymorphisms can affect the expression of CAT mRNA in tumor tissues[CAT expression is significantly longer (p = 0.020, p = 0.048 respectively; CAT expression in tumor cells, thus regulating ROS and making tumor cells sensitive to platinum-chemotherapy, but the specific mechanism needs to be further explored. Therefore, CAT can be used as a potential target to enhance the sensitivity of platinum-chemotherapy, nanocarriers of platinum and CAT enhance the cytotoxicity of drug resistant cancer cells[CAT expression (CAT genes. In conclusion, our study showed that CAT rs769217 is significantly related to PSF of platinum-based chemotherapy in lung cancer patients. CAT rs769217 may be a biomarker for predicting the prognosis of lung cancer patients with platinum-based chemotherapy.Our results suggest that otherapy . CAT genve stress,42. Prevve stress–45. Two te cancer,47. At tte cancer–50. Therell lines. But ther tissues, and acccer cells. In addiression . The exp


Regulated cell death (RCD) is a regulable cell death that involves well-organized signaling cascades and molecular mechanisms. RCD is implicated in fundamental processes such as organ production and tissue remodeling, removing superfluous structures or cells, and regulating cell numbers. Previous studies have not been able to reveal the complete mechanisms, and novel methods of RCD are constantly being proposed. Two metal ions, iron (Fe) and copper (Cu) are essential factors leading to RCDs that not only induce ferroptosis and cuproptosis, respectively but also lead to cell impairment and eventually diverse cell death. This review summarizes the direct and indirect mechanisms by which Fe and Cu impede cell growth and the various forms of RCD mediated by these two metals. Moreover, we aimed to delineate the interrelationships between these RCDs with the distinct pathways of ferroptosis and cuproptosis, shedding light on the complex and intricate mechanisms that govern cellular survival and death. Finally, the prospects outlined in this review suggest a novel approach for investigating cell death, which may involve integrating current therapeutic strategies and offer a promising solution to overcome drug resistance in certain diseases.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01267-1. In multicellular organisms, the process of generating new cells and removing damaged or unwanted cells maintains a dynamic balance during the development and maintenance of homeostasis. Thus, cell death is essential for life. In 1972, Kerr et al. first proposed a new term ‘apoptosis’ and described its morphological features, which has been a classic pattern of programmed cell death .To date, several studies have linked iron (Fe) and copper (Cu) with multiple forms of RCD. Newly discovered RCDs, ferroptosis and cuproptosis are dependent on these two transition metal ions respectively, which distinguish them from other RCDs , 5. FerrTo determine the effects of Fe and Cu on RCDs, we summarized almost all possible modes of iron and copper to impart significant cell damage and elaborated the mechanisms of iron- and copper-mediated diverse RCDs, including ferroptosis, cuproptosis, apoptosis, autophagy, necroptosis and pyroptosis Fig. . In addi2+, Cu 1+) and oxidized  states to perform numerous biological functions, including redox reactions, electron transport, oxygen transport and energy metabolism [Iron and copper belong to the first series of transition metals, which also includes chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni) and zinc (Zn). Iron and copper have similar characteristics, are both essential nutrients, are involved in fundamental biological processes and play a crucial role in health and disease , 9. In ltabolism –12.Iron and copper can also influence the functions of proteins upon binding to these proteins. On the one hand, Fe and Cu must be bound and protected within the active sites of proteins . For exaThe structure of the tyrosinase active site consists of a dual-core copper center and histidine residues . TyrosinIn addition, their relationship is close. Copper may positively influence the transport of iron. Systemic copper deficiency blocks iron transport and accumulates in tissues, ultimately generating cellular iron deficiency . ConversIt should be mentioned that everything has both sides. Iron and copper are beneficial to life in moderate amounts, but excesses or deficiencies of these metal ions are harmful. On the one hand, when there is too much iron in the body, excess iron forms a labile iron pool, confers cell toxicity, and affects cell damage, which can lead to cancer, hematological diseases, brain injury and other chronic and commonly encountered diseases . In addiIron and copper have many of the same or different modes that stimulate a range of indirect negative effects, provoking cell impairment and eventually cell death . Then, wOxidative stress is a state of oxidation-antioxidant imbalance and generates large amounts of reactive oxygen species (ROS). Moderate ROS levels assist in the control of cell proliferation and differentiation, but high concentrations of ROS are harmful to normal and cancer cells , 27. Whe2+ and Cu+ transform hydrogen peroxide into hydroxyl radicals (OH⋅). OH⋅ is one of the most reactive species found in nature and is extremely toxic.+ + H2O2 → Cu2+ + OH− + OH⋅Cu−based Fenton reaction Cu2+ + H2O2 → Fe3+ + OH− + OH⋅Fe−based Fenton reaction FeThe first mechanism by which Fe and Cu induce oxidative stress is through the Fenton reaction , 33, 37.The second way of inducing oxidative stress is the consumption of antioxidants. There are two kinds of antioxidants. One class of them is low-molecular-weight antioxidants, including glutathione (GSH), ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), carotenoids, flavonoids, and other antioxidants that are capable of chelating metal ions to inhibit their catalytic activity and reduce ROS . The othEvidence of copper and iron leading to antioxidant deficiency can be clearly seen in the case of GSH. GSH plays a critical role in removing ROS and is a substrate for multiple enzymes that remove ROS. Copper and iron can catalyze GSH oxidation, which oxidizes reduced GSH to oxidized glutathione disulfide (GSSG) to reduce the concentration of GSH . The depSome studies have revealed that transition metal ions, including Cu, Fe, Aurum (Au) and Manganese (Mn), can inhibit the proteasome , 41, 42.Iron has a separate function driving lipid peroxidation, while copper has separate functions, including breaking DNA directly, protein ubiquitination and driving protein lipoylation and aggregation Table .Table 2ICopper could influence DNA indirectly through ROS. Meanwhile, copper could also act on DNA directly in another way.Copper could bind to DNA to form a bis- copper(II) complex, which may be inserted into the minor groove in the DNA. This DNA–copper complex is oxidized in the presence of an activator, especially hydrogen peroxide. Then, hydrogen bonds of the DNA–copper complex are hydrolyzed, and DNA is cleaved , 56. AnoIron complexes that could bind and damage DNA were undetected. Instead, iron even assists DNA synthesis and repair and works as a cofactor of multiple enzymes, including multiple DNA repair enzymes  and ribonucleotide reductase .In addition to regulating the proteasome, copper also has another way to impact protein degradation. Recent work detected that Cu+ promoted multiple proteins ubiquitination and degradation by positive allosteric activation of the E2 conjugating enzyme clade UBE2D1–UBE2D4 . SeveralStudies have indicated that Cu(II) is effective at aggregating proteins . MeanwhiIt has been presented previously that aggregating proteins are not critical for Cu cytotoxicity. In vitro, Cu(II) caused bovine albumin (BSA) to aggregate in a time- and concentration-dependent manner. It is possible that this aggregation is the result of covalently cross-linked multimers because Cu(II) is able to oxidize –SH groups in different molecules. In addition, Cu(II) plays an important role in the nonamyloid aggregation of HγD crystallin in cataract disease. Copper ions alter the conformation of hexagonal D-crystallin, causing light-scattering aggregates with high molecular weights. Surprisingly, the interaction of Cu ions with HγD crystallin also promotes protein folding . In otheNonetheless, Tsvetkov et al. found that Cu drives protein lipoylation and aggregation, resulting in proteotoxic stress and further inducing cell death . This ilIn ferroptosis, overloaded iron(II) produces a large number of ROS via Fenton reaction, promotes the production of lipid ROS, and then promotes ferroptosis . SpecifiIron and copper mediate many forms of cell death. One or both metals could induce ferroptosis, cuproptosis, apoptosis, autophagy, necroptosis and pyroptosis in different ways Table .Table 3IIron and copper are able to induce ferroptosis , 6. Ferr3+, free Fe2+ ions show higher toxicity to induce ferroptosis. Fe3+-Compared to Fe3+ is reduced to Fe2+ by STEAP3 , and then Fe2+ is released into the cytosol through divalent metal transporter 1 (DMT1) [2+ forms the labile iron pool (LIP), which plays a significant role in ferroptosis [The transferrin (TF) complex binds with the membrane protein TFRC (transferrin receptor) and is imported into cells through endocytosis . In endo1 (DMT1) . Excess roptosis . In addiroptosis .Generally, cells manage iron through absorption, output, utilization, and storage . When inPolyunsaturated fatty acids (PUFAs), such as adrenoyl (ADA) and arachidonoyl (AA), are the easiest lipids to oxidize in ferroptosis . With th− is a membrane transport protein and consists of SLC7A11 and SLC3A2. System Xc− transports glutamate outside and cystine inside cells at a ratio of 1:1 [System Xco of 1:1 . Cystineo of 1:1 . Hence, 2+ directly binds to the C107 and C148 cysteine residues of the GPX4 protein, which induces GPX4 protein aggregation. Then, the aggregates are recognized by the autophagic receptor TAX1BP1 (Tax1 binding protein 1) and degraded by the autophagy pathway. Subsequently, ferroptosis occurs in response to autophagy [In general, Fe is the ferroptosis-inducing factor. Interestingly, a study found that Cu also induced ferroptosis . Gao et utophagy .Tsvetkov et al. discovered a new form of RCD termed cuproptosis . They reThe copper transporters SLC31A1 (CTR1), ATP7A and ATP7B are responsible for regulating the level of copper in cells . SLC31A1In addition, there are useful tools that can bind to copper ions and help them enter cells, named copper ionophores . These iGSH binding to copper is an important way to reduce copper accumulation. Tsvetkov et al. also found that the GSH synthase inhibitor BSO induced cuproptosis.The TCA cycle can control cell fate and function, serving as an important route for oxidative phosphorylation and regulating redox, biosynthetic and bioenergetic balance . TsvetkoLipoylation is a posttranslational modification in which lipoic acid is attached to proteins. This modification is unique to the pyruvate dehydrogenase complex (PDC) in the TCA cycle . In mitoFDX1 is a mitochondrial reductase that reduces Cu2+ to its cuprous and more toxic form, Cu1+. In addition, FDX1 binds and is inhibited by elesclomol . HoweverAlthough ferroptosis inhibitors do not inhibit copper-induced cell death in cells, excessive copper leads to a loss of Fe-S cluster proteins under the regulation of FDX1  Fig. 3)3). TheseIn addition, GSH is a known copper and iron chelate that can reduce metal ion toxicity. GSH serves as the principal substrate for GPX4 and acts as a suppressor of ferroptosis. The induction of cuproptosis by the GSH synthase inhibitor BSO shows that GSH serves the same function in cuproptosis. These observations raise the possibility of crosstalk between Cu and Fe.Apoptosis is a classic RCD that can be induced by both external and internal factors. External apoptosis is initiated by cell membrane proteins known as death receptors . IntrinsThere are a number of reports about Fe- and Cu-mediated apoptosis Fig. A. In thoIron and copper can trigger intrinsic apoptosis. In osteoblasts, iron overload induces intrinsic apoptosis via ROS. Tian et al. noted that excess iron effectively causes apoptosis in osteoblasts in vitro. Iron overload leads to an increase in ROS that decreases and depolarizes MMP, increases Bax and cleaved caspase-3, and reduces Bcl-2. Changes in Bcl-2 and Bax expression also promote mitochondrial membrane permeability . In addi2+ can also modify the permeability of mitochondrial membranes, cause mitochondrial membrane depolarization, decrease mitochondrial membrane potential, reduce cytochrome c oxidase activity, and eventually induce intrinsic apoptosis [Cupoptosis , 66, 101poptosis . Rochforpoptosis .Except for intrinsic apoptosis, Fe and Cu also induce external apoptosis. Dehydroabietic acid (DHC)-Fe(III) and DHC-Cu(II) trigger mitochondrial intrinsic and extrinsic apoptosis. These complexes activate caspase-9, increase Bax, reduce Bcl-2 (intrinsic pathway), and facilitate caspase-8/caspase-4 and Fas (extrinsic pathway) . DHC- coSeveral studies have documented that iron mediates autophagy Fig. B, a procCu complexes also lead to autophagy , 73, 74.Fe also triggers necroptosis Fig. C, with fSeveral pathways contribute to pyroptosis, including the Caspase-1 activation-mediated classical pyroptotic pathway, Caspase-4/5/11-dependent nonclassical pyroptotic pathway, Caspase-8-dependent pyroptotic pathway and Caspase-3-dependent pyroptotic pathway .It is now well established from several studies that iron is a crucial inducer of pyroptosis Fig. D. Zhou eCopper can also induce pyroptosis by relying on ROS accumulation . In hepaSeveral types of cell death, including apoptosis, autophagy, necroptosis, pyroptosis and cuproptosis, are related to ferroptosis. However, the cross between cuproptosis and other forms of RCD has not been reported.Ferroptosis is associated with apoptosis. p53 participates in the regulation of both ferroptosis and apoptosis. P53 is an important regulator of apoptosis, and a large number of apoptotic factors are dependent on P53 activation to regulate apoptosis. In addition, p53 can trigger apoptosis by provoking mitochondrial translocation and accelerating cytochrome c release directly . p53 alsOther studies have shown that autophagy also plays a role in the occurrence of ferroptosis , 115. AcNecroptosis and ferroptosis often co-occur in diverse diseases. In hemorrhagic stroke, ferroptotic (activating phospho-ERK1/2) and necroptotic cell death (increasing RIP1 and RIP3 mRNA expression and activating phospho-RIP1) simultaneously occur . Basit ePyroptosis and ferroptosis often occur simultaneously in diverse diseases as well. In colorectal cancer, NFS1 knockout combined with oxaliplatin causes PANoptosis  by increasing ROS . PyroptoFerroptosis is also related to cuproptosis. It was recently revealed that the ferroptosis inducers sorafenib and erastin promote cuproptosis by enhancing copper-dependent lipoylated protein aggregation in primary liver cancer cells . In a luAlthough none of the studies reviewed the cross between cuproptosis and apoptosis, autophagy, necroptosis and pyroptosis, some clues offer the possibility. A review summarized the promotion effects on RCDs of tumor suppressor p53, including apoptosis, ferroptosis, parthanatos, programmed necrosis, and autophagic cell death. They stated that p53 might also play a role in cuproptosis because the gene regulates the biogenesis of iron-sulfur clusters and the copper chelator glutathione, which are two critical components of the cuproptotic pathway . AnotherBased on the importance of copper and iron in diseases, a multitude of strategies have been developed to regulate intracellular copper and iron levels. One of the major roles of those agents is developing novel anticancer therapies. Iron and copper are vital for tumorigenesis and cancer progression. In particular, cancer cells have been shown to have higher iron requirements than normal cells, often referred to as ‘iron addiction’ . Two polIron and copper targeting strategies have been used extensively Table . For exaIron (Fe) and copper (Cu) are the first series of transition metals that are essential nutrients, are involved in fundamental biological processes and play a crucial role in health and disease. Moderate Fe and Cu are beneficial to life, but excesses or deficiencies are harmful. Therefore, Fe and Cu are sometimes toxic to cells. Iron and copper own or co-own modes that lead to cell impairment and eventually cell death.In this review, we described some modes of iron and copper that may be deleterious to cell growth directly or indirectly. We found that iron and copper are able to impair cells through excessive ROS and proteasome inhibition. In addition, iron can drive lipid peroxidation, which leads to ferroptosis, while copper can bind and break DNA and bind and activate E2D2-inducing protein ubiquitination and degradation.and drive protein lipoylation and aggregation, inducing cuproptosis as well.Notably, it is important to tell the real reason for DNA damage: iron and copper influence ROS, evocating DNA damage indirectly, or copper binds and breaks DNA directly. In addition, it is also noteworthy to judge whether protein ubiquitination induced by copper harms cells. Copper promotes target polyubiquitination and can thus regulate the degradation rate of many proteins that are also favorable for cell growth. For example, protein ubiquitination induced by copper is helpful in development and head formation in Drosophila . Cu+ canIron and copper mediate diverse forms of cell death. Both Fe and Cu can induce extrinsic and intrinsic apoptosis, autophagy, ferroptosis and pyroptosis. Only iron is capable of inducing necroptosis, while copper is able to trigger cuproptosis. From the studies discussed thus far, copper has more functions to induce cell death, which acts on proteins compared to iron. We hypothesize that copper may also trigger necroptosis or ferroptosis by impacting protein structure and function, which may provide a new research direction.Types of cell death may be related to one another, and iron- and copper-mediated cell death is likely to be interrelated as well. Ferroptosis, as an independent mode of cell death, is related to apoptosis, autophagy, necroptosis, pyroptosis and cuproptosis, which have been extensively reported. However, only very few findings have offered clues regarding the cross between cuproptosis and other forms of RCD. There are several ways of cross talk between RCDs. First, causal relations between autophagy and ferroptosis, ferroptosis and necroptosis and ferroptosis and cuproptosis are present. Two different types of autophagy, ferritinophagy and lipophagy, producing excessive free iron ions and fatty acids, respectively, result in ferroptosis. Ferroptosis contributes to the occurrence of necroptosis. Ferroptosis inducers and regulatory genes also contribute to the occurrence of cuproptosis. Second, there are some common points between apoptosis and ferroptosis, pyroptosis and ferroptosis and cuproptosis and multiple RCDs. IFN-γ works as both apoptosis- and ferroptosis-inducing factors. Pyroptosis and ferroptosis have the same point, lipid peroxidation. p53 has promoting effects on RCDs, including apoptosis, ferroptosis, cuproptosis, and autophagic cell death. HMGB1 also plays an important role in cuproptosis and autophagy. Third, necroptosis, pyroptosis and ferroptosis, as well as ferroptosis and cuproptosis, often cooccur in diverse diseases. Fourth, pyroptosis also acts cooperatively with ferroptosis to lead to many diseases. The cross-talk between ferroptosis and cuproptosis with other cell death provides the possibility of joint application of existing treatment schemes and helps to solve drug resistance issues in some diseases, which may provide a new research direction.Cell death has advantages and disadvantages for individuals. On the one hand, the low viability of normal cells compromises individual survival. On the other hand, tumor cell death is beneficial to life prolongation. In this study, we described iron- and copper-induced cell death. Saving normal cells alive or killing cancer cells by regulating iron- and copper-mediated cell death may be a smart approach. In addition, the pattern and relationships between ferroptosis and other forms of death involvement in diseases determine the drugs that can be adopted to prevent uncontrolled cell death.Does copper also contribute to necroptosis? What is the relationship between cuproptosis and different types of cell death? Is it synergy or antagonism? Whether similarities and differences between copper and iron can help us explore the detailed mechanisms of cell death mediated by them. Whether these various modes of cell death can be integrated into a complete regulatory network still requires further exploration.


Do patients with breast cancer tend to discontinue their cardiovascular therapy at the same time they discontinue adjuvant hormone therapy?In this population-based cohort study of 5493 patients, the highest risk of discontinuing cardiovascular therapy was observed at the time when patients discontinued adjuvant hormone therapy. Furthermore, patients who discontinued adjuvant hormone therapy were at a higher risk of both breast cancer mortality and cardiovascular mortality.These findings suggest that multiple disease−focused interventions are warranted to prevent discontinuation of treatment for other diseases and to improve overall survival in patients with breast cancer. This cohort study investigates whether patients with breast cancer who discontinue adjuvant hormone therapy tend to discontinue their cardiovascular therapy concomitantly, examines disease-specific morality by discontinuation of adjuvant hormone therapy, and identifies factors associated with discontinuation of therapy among patients with breast cancer. A large proportion of patients with breast cancer concomitantly use adjuvant hormone therapy and cardiovascular therapy.To examine the relative risk of discontinuing cardiovascular therapy during the periods before and after discontinuation of adjuvant hormone therapy.This population-based cohort study included all women aged 40 to 74 years in Stockholm, Sweden, who were diagnosed with breast cancer and concomitantly using adjuvant hormone therapy and cardiovascular therapy. Patients were enrolled from July 1, 2005, to August 31, 2020, with a median follow-up of 7.2 years. Data were analyzed from November 3, 2021, to May 12, 2022.Discontinuation of adjuvant hormone therapy.The main outcome was discontinuation of cardiovascular therapy  within 1 year before and after discontinuation of adjuvant hormone therapy. Incidence rate ratios with 95% CIs were estimated using Poisson regression. Furthermore, hazard ratios (HRs) with 95% CIs for cause-specific mortality were estimated using Cox proportional hazards regression models, comparing those who discontinued and continued adjuvant hormone therapy.A total of 5493 patients with breast cancer who concomitantly used cardiovascular therapy were identified; 1811 who discontinued adjuvant hormone therapy were individually matched to 1 patient each who continued therapy by year of breast cancer diagnosis, age at diagnosis, and use of the same cardiovascular therapy. Most patients (4070 [74.1%]) were aged 60 years or older at diagnosis. At the time when patients discontinued adjuvant hormone therapy, 248 (12.2%) concomitantly discontinued their cardiovascular therapy. During follow-up, a higher discontinuation rate of cardiovascular therapy was also observed among those who discontinued adjuvant hormone therapy. Consistently, adjuvant hormone therapy discontinuation was associated with an increased risk of death not only due to breast cancer  but also cardiovascular disease . Stratifying the analyses on baseline type of adjuvant hormone therapy yielded consistent results.In this cohort study of data from population-based registers in Sweden, patients who discontinued adjuvant hormone therapy were also more likely to discontinue cardiovascular therapy, especially at the time when they discontinued adjuvant hormone therapy. These findings suggest that clinicians should shift from single- to multiple-disease focus to prevent discontinuation of therapies for other diseases among patients with breast cancer. However, nonadherence to long-term cardiovascular therapy remains a concern for the expanding population of patients who survive breast cancer.18 Despite previous studies having identified a few shared risk factors for discontinuation of adjuvant hormone therapy and cardiovascular therapy,15 little is known about whether discontinuation of these 2 therapies happens simultaneously.Improved survival in patients with breast cancer has brought increased incidence of comorbidities. Cardiovascular disease is one of the most frequent comorbidities among patients with breast cancer.We hypothesized that patients who discontinue adjuvant hormone therapy were more likely to discontinue cardiovascular therapy and thus had higher mortality due to cardiovascular disease. Specifically, our study aims to (1) investigate whether patients with breast cancer who discontinue adjuvant hormone therapy tend to discontinue their cardiovascular therapy concomitantly; (2) examine breast cancer–specific mortality and cardiovascular disease–specific morality by discontinuation of adjuvant hormone therapy; and (3) identify shared and unshared factors associated with discontinuation of adjuvant hormone therapy and cardiovascular therapy.STROBE) reporting guideline.The regional Ethical Review Authority in Stockholm, Sweden, approved this cohort study. Informed consent is not required for large-scale, registry-based studies in Sweden, provided that the study is deemed ethical by the Ethical Review Authority. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology ,24 Multi-Generation Register,25 National Cancer Register,26 and Cause of Death Register.27 The Stockholm-Gotland Quality Register for Breast Cancer  includes all patients with breast cancer diagnosed in the Stockholm-Gotland region and provides detailed information on tumor characteristics and treatments, with 99.9% of incident breast cancer cases being recorded.20 The Prescribed Drug Register contains prescription records nationwide since July 2005, with 99.7% completeness.21 The Cause-of-Death Register (1952 onward) assesses the underlying cause of death according to strict rules by the patient’s usual physician or the physician last seeing the patient before death and records a specific underlying cause of death for 96% of individuals.27Using unique personal identification numbers,Through linkage between the Stockholm-Gotland Quality Register for Breast Cancer and the Prescribed Drug Register, we identified 13 053 women diagnosed with nonmetastatic and estrogen receptor–positive breast cancer at 40 to 74 years of age from July 1, 2005, to August 31, 2020, in Stockholm, Sweden, who initiated adjuvant hormone therapy  code L02BA01] and/or aromatase inhibitors [ATC code L02BG]). Among them, we further identified 5493 patients with breast cancer who concomitantly used cardiovascular therapy, defined as filling at least 2 prescriptions of cardiovascular drugs , statins (ATC code C10AA), or aspirin (ATC code B01AC06) after adjuvant hormone therapy initiation and before end of follow-up , death, emigration, completion of 5-year adjuvant hormone therapy, or end of the study period , whichever came first. Analyses for cardiovascular therapy discontinuation were additionally censored at occurrence or recurrence of cardiovascular comorbidities after start of follow-up, including heart failure , arrhythmias (ICD-10 codes I47-I49), ischemic heart disease (ICD-10 codes I20-I25), and cerebrovascular diseases (ICD-10 codes I60-I69).Discontinuation of adjuvant hormone therapy and cardiovascular therapy was defined as failure to refill a prescription of the corresponding therapy within 6 months after a previous prescription.ICD-10 code C50 and death due to cardiovascular disease through ICD-10 codes I00 to I99 in the Cause-of-Death Register. Patients were followed up until death, emigration, or end of the study period , whichever came first. The median follow-up time was 6.5 years.The main underlying cause of death was used to identify death due to breast cancer through 24 Use of symptom-relieving drugs, including analgesics (ATC code N02), gastrointestinal tract drugs , antidepressants (ATC code N06A), or hypnotics and/or sedatives (ATC code N05C), was defined as having 1 prescription of corresponding drugs during the first 6 months of adjuvant hormone therapy. Prediagnosis hormone replacement therapy was defined as a systematic use of estrogen and/or progesterone  with at least 1 prescription within 6 months before breast cancer diagnosis, excluding patch and vaginal cream preparations. Participation in mammography screening was defined as attendance of mammography within 2 years before breast cancer diagnosis. Family history of breast cancer and major cardiovascular events was obtained by linking the Multi-Generation Register to the National Cancer Register and National Patient Register, respectively. Charlson Comorbidity Index score before breast cancer diagnosis was calculated using main diagnoses retrieved from the Swedish Patient Register.28Information on divorce and income at diagnosis was retrieved from the LISA register.29Data were analyzed from November 3, 2021, to May 12, 2022. To investigate whether adjuvant hormone therapy and cardiovascular therapy tend to be discontinued concomitantly, we first identified 1811 patients who discontinued adjuvant hormone therapy and used cardiovascular therapy within 1 year before the therapy discontinuation. We compared the discontinuation rate of cardiovascular therapy at adjuvant hormone therapy discontinuation with other periods . Poisson regression with a clustered sandwich estimator was used to account for lack of independence due to patients using more than 1 cardiovascular therapy.2 tests to compare baseline characteristics between those who continued and discontinued adjuvant hormone therapy. Poisson regression with clustered sandwich estimator was used to estimate the association, adjusting for matching variables, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. This analysis was stratified by 4 time bands in relation to adjuvant hormone therapy discontinuation: −12 to −3 months, −3 months, 3 months, and 3 to 12 months.To further investigate the association between discontinuation of adjuvant hormone therapy and cardiovascular therapy, we used a matched cohort. For each patient who discontinued adjuvant hormone therapy, we randomly sampled 1 patient who continued the therapy at the first patient’s discontinuation date and used the same cardiovascular medication. Those who discontinued and continued medications were individually matched on year of breast cancer diagnosis and age at diagnosis (±3 years). We used χKaplan-Meier curves were further constructed for breast cancer–specific and cardiovascular disease–specific mortality for those who discontinued adjuvant hormone therapy and matched patients who continued therapy. Marginal Cox proportional hazards regression models with clustered sandwich estimator were used to compare cause-specific mortality between patients who discontinued and matched patients who continued therapy, adjusting for matching variables, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. All the aforementioned analyses were stratified by baseline type of adjuvant hormone therapy.Finally, we identified shared and unshared factors associated with discontinuation of adjuvant hormone therapy and cardiovascular therapy among patients who concomitantly used these 2 therapies. Marginal Cox proportional hazards regression models with clustered sandwich estimator were used, adjusting for age at breast cancer diagnosis, diagnosis calendar period, tumor size, lymph node status, tumor grade, progesterone receptor status, surgery type, chemotherapy, radiotherapy, baseline hormone therapy type, prediagnosis use of cardiovascular therapy, prediagnosis major cardiovascular event, and Charlson Comorbidity Index score at diagnosis. All analyses were performed using SAS, version 9.4 (SAS Institute Inc); Stata, version 17.0 (StataCorp LLC); or R, version 1.4.1106  at a 2-tailed α = .05.Of the 13 053 patients who initiated adjuvant hormone therapy, 5493 (42.1%) concomitantly used at least 1 of the following cardiovascular therapies: 4966 (38.0%) used cardiovascular drugs, 1861 (14.3%) used statins, and 968 (7.4%) used aspirin. In the full cohort of patients who concomitantly used cardiovascular therapies, 4070 patients (74.1%) were diagnosed after 60 years of age and 4046 of 5176 (78.2%) used the corresponding cardiovascular therapy before breast cancer diagnosis . Table 1At discontinuation of adjuvant hormone therapy, 248 of 2030 patients (12.2%) also discontinued their cardiovascular therapy . The discontinuation rate of cardiovascular therapy was the highest at the time of discontinuing adjuvant hormone therapy compared with other periods of follow-up, with an indcidence rate ratio (IRR) of 197.63 . Stratified analyses by baseline type of adjuvant hormone therapy yielded consistent results, with IRRs of 199.35  and 194.81  for aromatase inhibitors users and tamoxifen users, respectively.During the period 1 year before and after discontinuation of adjuvant hormone therapy, the incidence of discontinuing cardiovascular therapy was 0.82 per 1000 person-days among discontinuers and 0.41 per 1000 person-days among continuers. Discontinuers were more likely to discontinue cardiovascular therapy, with IRRs of 1.83  and 2.31  within 3 months before and after discontinuation, respectively . SimilarOur findings suggest that patients with breast cancer who discontinued adjuvant hormone therapy were also more likely to discontinue their cardiovascular therapy. In particular, patients with breast cancer tended to discontinue these 2 therapies at the same time. Moreover, those who discontinued adjuvant hormone therapy were at a higher mortality risk due not only to breast cancer but also cardiovascular disease.30 Elderly patients now account for most patients who survive breast cancer, and the number is projected to double by 2040.32 Such a large number highlights the clinical importance of monitoring use of cardiovascular medications among patients with breast cancer.Our findings further suggest that more than 2 in 5 patients with breast cancer concurrently used cardiovascular therapy and adjuvant hormone therapy. Many patients with breast cancer, especially elderly patients, present with multiple chronic diseases and concomitantly use several long-term medications.We also found that among concomitant users of adjuvant hormone therapy and cardiovascular therapy, the discontinuation of these 2 therapies tends to happen concomitantly. More than 1 in 9 patients with breast cancer discontinued their cardiovascular therapy when they discontinued their adjuvant hormone therapy. This finding suggests that, once oncologists detected discontinuation of adjuvant hormone therapy, they should ask patients about their adherence to cardiovascular therapy. That is, oncologists should shift from single- to multiple-disease focus to prevent discontinuation of treatment by patients with breast cancer, which is likely to be achieved through an extended cardio-oncology approach and cooperation.34 showing that nonadherence to cardiovascular therapy before breast cancer diagnosis is associated with nonadherence to adjuvant hormone therapy.In addition to concomitant discontinuation, we found that patients who discontinued adjuvant hormone therapy were also more likely to discontinue cardiovascular therapy in other time periods, both before and after adjuvant hormone therapy discontinuation. This result is consistent with results of a previous study36 indicating that nonadherence to cardiovascular medications following breast cancer diagnosis is associated with a higher level of low-density lipoprotein and an increased risk of a cardiac event. To potentially prevent these unnecessary deaths, interventions focusing on multiple medications adherence are urgently needed. Furthermore, while the association with cause-specific mortality did not significantly differ by hormone therapy type, it is important for future studies with a larger sample size to replicate this analysis considering the difference in efficacy and adverse effects profile between tamoxifen and aromatase inhibitors.38As expected, we found that adjuvant hormone therapy discontinuation was associated with an increased mortality not only due to breast cancer but also cardiovascular disease, after adjusting for tumor characteristics, treatments, prediagnosis major cardiovascular event, and prediagnosis use of cardiovascular therapy. Our result suggests that the concomitant discontinuation is highly likely to translate to higher mortality. This aligns with previous studiesDiscontinuation of adjuvant hormone therapy and cardiovascular therapy may share common risk factors and underlying mechanisms. In our study, we found that nonparticipation in mammography screening, divorce, low income, and use of symptom-relieving drugs are associated with both discontinuation of adjuvant hormone therapy and discontinuation of cardiovascular therapy. These shared factors may serve as potential targets for interventions that may simultaneously improve adherence to more than 1 life-saving medication, thus having greater cost-effectiveness. For example, we found that patients who used symptom-relieving drugs were at higher risk of discontinuing both therapies, suggesting that only prescribing symptom-relieving drugs seems to be not enough to prevent therapy discontinuation once patients have therapy-related symptoms. Other additional interventions, such as better clinician-patient communication, are needed to improve adherence for both adjuvant hormone therapy and cardiovascular therapy.Despite that, we also identified several unique factors specifically associated with discontinuation of adjuvant hormone therapy or discontinuation of cardiovascular therapy. For example, women with a family history of breast cancer were less likely to discontinue their adjuvant hormone therapy, but no difference was found for discontinuation of cardiovascular therapy. These unique factors suggest that discontinuation of different therapies also has its own unique mechanism and thus should be investigated separately to build a full picture of discontinuation of specific therapy.This study has several limitations. First, using prescription data from the register may underestimate the discontinuation rate, because prescription refill does not necessarily guarantee that a patient used the drug. Second, misclassifying patients who used aspirin as nonusers is possible because the Swedish Prescribed Drug Register does not capture over-the-counter medications. However, given the fact that low-dose and/or long-term use of aspirin is prescribed, we anticipate this misclassification is likely minimal. Third, although we censored patients at disease progress of both breast cancer and cardiovascular disease and adjusted for Charlson Comorbidity Index score at diagnosis and cancer treatments, we cannot rule out the possibility that the concomitant discontinuation of adjuvant hormone therapy and cardiovascular therapy could have been a result of clinical decision-making. While this may partially contribute to the observed association in our study, it is unlikely to substantially account for all associations we have reported. Fourth, the study did not have sufficient power to conduct a stratification analysis by timing of initiating cardiovascular therapy due to limited sample size for patients who initiated cardiovascular therapy after breast cancer diagnosis. Fifth, caution is needed when generalizing our findings to other countries, since our study population included only patients with breast cancer diagnosed in Sweden.In this cohort study of patients with breast cancer concomitantly using adjuvant hormone therapy and cardiovascular therapy, our results call for extra vigilance for concomitant discontinuation of adjuvant hormone therapy and cardiovascular therapy in clinical practice. Shared risk factors identified in our study may help to develop targeted interventions to simultaneously improve adherence to both therapies and subsequently improve overall survival among patients with breast cancer.


Introduction: A soft pneumatic muscle was developed to replicate intricate ankle motions essential for rehabilitation, with a specific focus on rotational movement along the x-axis, crucial for walking. The design incorporated precise geometrical parameters and air pressure regulation to enable controlled expansion and motion.Methods: The muscle’s response was evaluated under pressure conditions ranging from 100-145 kPa. To optimize the muscle design, finite element simulation was employed to analyze its performance in terms of motion range, force generation, and energy efficiency. An experimental platform was created to assess the muscle’s deformation, utilizing advanced techniques such as high-resolution imaging and deep-learning position estimation models for accurate measurements. The fabrication process involved silicone-based materials and 3D-printed molds, enabling precise control and customization of muscle expansion and contraction.Results: The experimental results demonstrated that, under a pressure of 145 kPa, the y-axis deformation (y-def) reached 165 mm, while the x-axis and z-axis deformations were significantly smaller at 0.056 mm and 0.0376 mm, respectively, highlighting the predominant elongation in the y-axis resulting from pressure actuation. The soft muscle model featured a single chamber constructed from silicone rubber, and the visually illustrated and detailed geometrical parameters played a critical role in its functionality, allowing systematic manipulation to meet specific application requirements.Discussion: The simulation and experimental results provided compelling evidence of the soft muscle design’s adaptability, controllability, and effectiveness, thus establishing a solid foundation for further advancements in ankle rehabilitation and soft robotics. Incorporating this soft muscle into rehabilitation protocols holds significant promise for enhancing ankle mobility and overall ambulatory function, offering new opportunities to tailor rehabilitation interventions and improve motor function restoration. Assistive robots have long been introduced into the field of rehabilitation. Despite the contentious progression of inflexible robots, these machines still suffer from numerous limitations that prove challenging to overcome. These limitations primarily stem from the materials utilized in their construction, which possess a substantial Young’s modulus, and the inherent constraints imposed by their structures. Simultaneously, rehabilitation exercises often require high flexibility and freedom of movement. Traditional rehabilitation exoskeletons, predominantly composed of rigid components such as linkages and hinges, tend to increase the weight borne by patients, thus inducing discomfort. Moreover, the attainment of multi-degree-of-freedom motion requires additional drive components and intricate structures . ConsequIn contrast to rigid rehabilitation robots, which have inherent limitations, soft robots offer distinctive advantages in terms of safety and adaptability. The abundance of degrees of freedom in soft robots effectively compensates for many of the limitations encountered with their rigid counterparts, allowing greater range of motion and versatility . Soft reComposed of flexible materials that mimic biological properties, soft rehabilitation robots offer high adaptability, flexibility, and safety, specifically regarding human-computer interaction and pneumatic-driven actuators . This deOver the years, artificial muscles have garnered increasing interest, with their initial prototypes dating back several years . One notA two-dimensional design of elastic muscles was proposed . In its A bubble artificial muscle (BAM) was proposed, which differs from the radial pleated muscle. This artificial muscle restricts its expansion by placing metal rings on plastic tubes, forming circumferential pleats, and dividing them into several units. This design achieves a similar contraction rate of 45%–50% . The RobIn the realm of rehabilitation technology, significant advancements have been made in the development of exoskeleton-assisted systems, particularly in the field of upper limb rehabilitation . One notFurthermore, recent developments have focused on incorporating synchronized fingertip haptic stimulation into exoskeleton-assisted hand rehabilitation systems. These innovative approaches have demonstrated promising results, particularly in enhancing attention levels and user engagement, especially when heavier grasping weights are involved. Including haptic stimulation has proven to be instrumental in improving rehabilitation outcomes and promoting active user participation . The colA comprehensive approach to wrist rehabilitation utilizes a compact and low-profile soft robotic wrist brace constructed from ethylene-vinyl acetate material. Integrating eight soft origami-patterned actuators onto a commercially available brace significantly enhances its functionality. Furthermore, the adoption of blow molding techniques enables cost-effective mass production of these actuators, ensuring the scalability and reproducibility of the device . A serieThe experimental analysis unveils noteworthy enhancements achieved by the robotic limb system compared to traditional hand drilling methods. Particularly significant is the reduction in vibration transmission by an impressive range of 40%–60%, all while maintaining satisfactory time performance. These findings underscore the potential of the developed system to enhance worker safety, minimize the incidence of injuries, and optimize workplace ergonomics and worker wellbeing . The comAn investigation introduces a wearable system that integrates various assistive technologies for individuals with upper-limb impairments . The sysA novel 4-degree-of-freedom (DOF) lower limb rehabilitation robot has been introduced, offering flexion/extension (F/E) training for three limb joints and adduction/abduction (A/A) training for the hip joint . This inA soft-pneumatic actuator-driven exoskeleton designed specifically for hip flexion rehabilitation is presented in this study. Comprehensive testing and evaluation have confirmed the effectiveness of the exoskeleton in assisting hip flexion movements, generating substantial torque, and reducing muscle effort. These findings underscore the exoskeleton’s potential as a valuable tool for facilitating efficient and effective hip flexion rehabilitation and alleviating muscle burden . In anotMoreover, a bio-inspired controller that leverages motor primitives has been proposed for a lower limb exoskeleton. This controller effectively compensates for torque deficiencies and accommodates variations in gait characteristics, thus enhancing motor performance and synchronization between the human and exoskeleton system. The effectiveness of the motor primitive-based controller has been demonstrated in addressing motor deficiencies during lower limb movements . A novelFocusing on the limitations of rigid rehabilitation robots and the advantages of soft robots in rehabilitation. Despite integrating sensor systems, rigid rehabilitation exoskeletons still pose safety risks due to their hardness, high density, and inability to deform in response to external forces. In contrast, soft rehabilitation robots, with their flexibility and adaptability, have found utility in specific rehabilitation applications. However, existing soft actuators, such as artificial muscles actuated pneumatically, are susceptible to damage and limitations in performance. Therefore, there is a need for improved soft actuators that overcome these drawbacks. The paper aims to explore advancements in wearable systems and lower limb rehabilitation robots, specifically focusing on ankle rehabilitation. The paper aims to propose a compact and low-profile soft robotic ankle brace and investigate the development of a supernumerary robotic limb to mitigate injuries and reduce the lower limb’s joint load. The goal is to enhance individuals’ functional abilities and improve rehabilitation outcomes through innovative technologies and approaches tailored to ankle rehabilitation.x-axis, as this movement is pivotal in supporting the patient’s walking ability. Incorporating the soft muscle into the rehabilitation process is expected to assist patients in regaining ankle mobility and enhancing their overall ambulatory function.In the human leg, four muscles are crucial in facilitating ankle rotation movement: Peroneus brevis, Peroneus longus, Extensor digitorum longus, and Tibialis anterior . HoweverX-axis, as exemplified in In this study, a novel soft muscle design is proposed to replicate the complex movements of the ankle joint. As illustrated in The present study employs a systematic approach for selecting geometrical parameters to achieve optimal performance of the soft pneumatic muscles. A rigorous benchmarking process was undertaken, considering various factors such as motion range, force generation, and energy efficiency. The resulting model showcases promising motions that hold potential applications in post-stroke patient rehabilitation and physical therapy settings. To gain a comprehensive understanding of the soft muscle’s behavior, an in-depth analysis was carried out. This analysis evaluated the muscle’s motion under different conditions, specifically on predetermined air pressure values. By examining the muscle’s response across various pressures, valuable insights were obtained regarding its performance characteristics. The selected geometrical parameters, critical to the soft muscle’s functionality, are visually illustrated in Do), chamber height (H), pitch (P), and chamber thickness (T), as outlined in The design represents a single chamber but also contains a multi-air pillow is better to mention it as a multi-air pillow constructed from a flexible elastomeric material, specifically silicone rubber. Instead of a traditional reinforcement layer, chamber rings provide structural integrity. The controlled expansion of the soft muscle is achieved through the pressurization of these chambers with air. Key parameters governing the behavior of the soft muscle include the chamber length (L), outer diameter . Where the only material constant required by the simulation software is C30.Here, For the purposes of simulating the model in this study, finite element analysis was performed using ANSYS. The prototype was subjected to simulations ranging from a pressure of 105 KPa to 145 KPa. Then, the material parameters derived from the curve fitting process were subsequently integrated into the FEA software (ANSYS) to establish the finite element analysis. Detailed information regarding the specific material parameters of the hyperelastic model can be found in z-axes and quantitatively assess the distribution of stress within the structure of the soft muscle. To achieve this objective, the muscle underwent a meticulous examination with a wide range of pressures applied, ranging from 105 to 145 kPa in increments of 5 kPa. The pressure application was thoughtfully implemented, selectively targeting the inner surfaces of the muscle while ensuring that the outer surfaces were maintained at a constant atmospheric pressure.This rigorous simulation study systematically evaluated the mechanical response and performance of a soft pneumatic muscle for ankle rehabilitation under various loading conditions. The primary objective of this comprehensive analysis was to determine the maximum deformation along the x, y, and The findings obtained from this extensive simulation analysis, as visually presented in Y-axis (Y-def) exhibited a substantial measurement of 165 mm, clearly indicating a significant elongation primarily occurring in the Y-direction.From this meticulous analysis, several notable findings have emerged, providing valuable insights into the performance of the soft muscle with respect to deformation and stress. Remarkably, at a pressure of 145 kPa, the deformation along the x and z-axes were negligibly small, measuring 0.056 mm and 0.0376 mm, respectively, indicating minimal elongation along these axes. This observation emphasizes the specificity of the soft muscle response to pressure actuation in the intended direction. In addition, the controllability of the muscle at different pressure values was evident, as the elongation range spanned from 0 mm to 165 mm. This remarkable adaptability and controllability enable precise regulation of the ankle’s rotational angle, thus accommodating individual patient needs and facilitating customized rehabilitation protocols.This outcome underscores the robustness and efficacy of the soft muscle design, particularly in facilitating ankle flexion and extension. On the contrary, the deformations along the The visual representation in The soft muscle fabrication process for ankle rehabilitation is based on advances in soft robotics technology and molding techniques. These methods have attracted significant attention and recognition across various areas, including medical devices, prostheses, and wearable exoskeletons, with various areas attracting this method. Soft muscles have inherent qualities of flexibility and adaptability, making them very promising for rehabilitation applications where patient comfort and safety are paramount. The main purpose of designing soft muscles for ankle rehabilitation is to create devices that faithfully replicate the behavior of natural muscles and tendons, providing the required strength and range of motion for ankle movements such as flexion, extension, inversion, and eversion.In order to achieve this goal, the correct selection and optimization of the shape and silicone material properties of the mold and silicone material are essential. Because of its elasticity, durability, and biocompatible properties, silicone is an ideal material for prolonged contact with the human body. In addition, silicone enables silicone to be easily molded into custom shapes, allowing soft muscle development to be tailored to an individual’s specific needs and anatomical structure. It also allows for customization. Incorporating air pillows into soft muscle designs precisely controls muscle expansion and contraction. By manipulating the air pressure in the soft muscle, the appropriate level of support or resistance can be imparted during ankle exercises, thus facilitating patient-specific rehabilitation protocols. Additionally, the presence of air pillows increases the comfort of soft muscles, allowing them to adapt to the wearer’s movement accurately. Furthermore, air pillows enhance its overall compliance, allowing it to seamlessly adjust to the wearer’s movements, minimizing any discomfort or inconvenience.The manufacturing process includes the use of silicone-based materials and the creation of a hollow soft muscle model using two distinct 3D printed molds, as shown in Based on previous experiments and empirical evidence, the most effective adhesive for bonding two silicone components was the silicone material itself, as shown in An experimental platform was developed and implemented with meticulous attention to detail to investigate the deformation of soft muscle during actuation with different pressures ranging from 100 to 145 (Kpa) for ankle rehabilitation. The experimental setup consisted of several critical components, each serving a specific purpose to ensure accurate and reliable results. The soft muscle was first securely fixed at one end in a 3D environment, allowing for unrestricted movement and deformation. This freedom of movement was essential for observing muscle behavior under different pressure conditions. To facilitate precise measurements of the muscle’s displacement, a 1 m × 1 m cubic cell was employed as the workspace, with a grid sheet positioned behind the muscle as a reference to ensure accurate displacement measurements throughout the experiment, as shown in To deliver pressurized air and secure the muscle in place, cantilever support was attached to the muscle and suspended at the top of the cubic cell. The cantilever support was designed to be dual-purpose, essential in securing the muscle and providing the necessary pressure head changes. The pneumatic system incorporated into the platform allowed for regulating the pressure and flow rates delivered to the muscle, enabling precise control over the muscle’s response to pressure head variations. Accurate capture of the muscle’s movement was essential for analyzing its behavior during the experiment. To accomplish this, a high-resolution RealSense D435i camera was employed to record video footage of the muscle’s displacement in the X, Y, and Z directions. The camera’s high resolution provided an accurate and comprehensive record of the muscle’s response under varying pressure conditions, which was synchronized with other experimental parameters using LabVIEW software. The camera was positioned at a fixed distance from the muscle. It was oriented perpendicular to the grid sheet to ensure accurate measurement of the muscle’s displacement in all three dimensions.Deep learning position estimation models were used to locate and measure the soft muscle end tip position x y z, marked with a red mark, to investigate the relationship between pressure head changes and soft muscle elongation for ankle rehabilitation. The algorithm defined displacement relative to the calibrated end tip’s first position. Before starting the measurement process, the end tip position was located using an initial calibration procedure. The observed position changes were then plotted with respect to the change of pressure, as shown in In the realm of artificial muscle systems research, a comprehensive comparison was conducted between McKibben artificial muscles, Festo pneumatics, and an actuator specially designed within this study. The evaluation focused on several critical parameters, including actuation speed, force generation, durability, ease of control, and fabrication. These factors are instrumental in determining the design, operation, and overall efficacy of artificial muscles, particularly in applications such as ankle rehabilitation.Actuation speed was a key consideration. The actuator developed in this study was engineered to respond swiftly to control inputs, mirroring the rapid actuation characteristic of McKibben muscles. In contrast, slower actuation speeds were observed in Festo actuators, a trait shaped by their distinct design and operational principles. In terms of force generation, McKibben artificial muscles have been widely recognized for their high force-to-weight ratio. The actuator developed in this study, while not precisely matching the force generation of McKibben muscles, was found to generate a substantial force aptly suited for the intended applications. Festo actuators, conversely, were found to offer a range of force generation capabilities, primarily determined by the specific model, but a high force output is generally provided.The durability of these artificial muscles was also examined. Both McKibben muscles and Festo actuators were found to exhibit robustness in a variety of applications. The actuator developed within this study, constructed from durable silicone and featuring an innovative design, also demonstrated promising durability, as evidenced by repeated testing cycles. Ease of control emerged as another vital parameter. The control of McKibben muscles can prove complex owing to their non-linear behavior. Festo actuators, despite being easier to control, often necessitate complex control systems. This complexity was mitigated in the developed actuator through design for easy control, a feature augmented by a LabVIEW-based control scheme.Fabrication was another domain where the developed actuator excelled. It was designed with a strong focus on simple fabrication and assembly, as detailed in this paper. In contrast, both McKibben and Festo actuators were found to require more complex fabrication and assembly processes, which can pose significant challenges to their production and implementation.Finally, McKibben artificial muscles and Festo pneumatic actuators each demonstrate their own strengths and have proven their effectiveness in various applications, the soft actuator developed in this study exhibits promising characteristics. These attributes position it as a potential candidate for targeted applications, particularly in the field of ankle rehabilitation.x-axis rotational movement during walking. The study achieved controlled expansion, accurate measurement, and comprehensive analysis of the muscle’s performance by integrating precise geometrical parameters, air pressure regulation, and advanced experimental techniques. Rigorous finite element simulation and experimental investigations provided compelling evidence of the soft muscle design’s adaptability, controllability, and effectiveness, establishing a solid foundation for future advancements in ankle rehabilitation and soft robotics. The experimental results revealed noteworthy findings. Under a pressure of 145 kPa, the soft muscle exhibited a substantial deformation along the y-axis (y-def) measuring 165 mm, emphasizing its significant elongation primarily in the y-direction. Conversely, the deformations along the x-axis and z-axis were minimal, measuring only 0.056 mm and 0.0376 mm, respectively, indicating negligible elongation along these axes. This observation underscores the specific and targeted response of the soft muscle to pressure actuation in the intended direction. Furthermore, the findings demonstrated the controllability of the muscle across different pressure values, showcasing an elongation range spanning from 0 mm to 165 mm. This remarkable adaptability and controllability enable precise regulation of the ankle’s rotational angle, accommodating the unique needs of individual patients and facilitating tailored rehabilitation protocols. The comprehensive understanding gained from this study significantly contributes to soft robotics and offers valuable guidance for future research and development in medical applications. The experimental platform developed for investigating the soft muscle’s deformation under various pressure conditions proved a reliable and accurate tool, enabling precise control and measurement of the muscle’s response. Overall, the results obtained from this study enhance our understanding of the intricate relationship between pressure head changes and soft muscle elongation for ankle rehabilitation. The measured values reinforce the performance and robustness of the soft muscle design, providing a solid foundation for further advancements in ankle rehabilitation and soft robotics. This research opens up new possibilities for improving ankle mobility and overall ambulatory function, leading to enhanced rehabilitation outcomes in clinical practice.In this research study, a soft pneumatic muscle was successfully developed and evaluated to replicate the intricate ankle motions necessary for effective rehabilitation, specifically emphasizing the crucial