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sampling for additional evidence of disease. often presents as a large, destructive mass. As with many other aggressive hematolymphoid neoplasms, however, it is potentially curable with appropriate chemotherapy, COMMON LYMPHOMAS AFFECTING THE most often with a combination of cyclophosphamide, vin- GASTROINTESTINAL TRACT cristine, doxorubicin, and dexamethasone (CHOP), and possibly with the addition of anti-CD20 immunotherapy While individual lymphomas are discussed and illustrated such as rituximab. in much more detail in Chapters 8, 9, and 11, several of the most commonly encountered entities are briefly reviewed here, followed by a general, pragmatic overview to lym- MALT Lymphoma phoma diagnosis and a handful of specific illustrative The correct term for “MALT” lymphoma is actually “extra- scenarios that raise points of importance and potential nodal marginal zone lymphoma of mucosa-associated TABLE 4.1 Antigen Expression Patterns of Various GI Lymphomas Antigens Lymphoma CD2 CD3 CD4 CD5 CD7 CD8 CD10 CD20 CD43 CD56 CD79a BCL-2 BCL-6 Cyclin-D1 PAX-5 DLBCL NH NH NH −/+ NH NH +/− + +/− − + + +/− − + MALT NH NH NH − NH NH − + −/+ − + + − − + FL NH NH NH − NH NH + + − − + + + − + MCL NH NH NH + NH NH − + + − + + − + + BL NH NH NH − NH NH + + − − + − + − + EATL + + − − + −/+ NH NH + − NH NH NH NH NH EATL-II + + − − + +/− NH NH + + NH NH NH NH NH ENKTL + − − − − − NH NH + + NH NH NH NH NH Abbreviations: BL, Burkitt lymphoma; DLBCL, diffuse large B cell lymphoma; EATL, enteropathy-associated T cell lymphoma; EATL-II, “type II” EATL; ENKTL, extranodal NK/T cell lymphoma, nasal type; FL, follicular lymphoma; MALT, mucosa-associated lymphoid tissue; MCL, mantle cell lymphoma; NH, not helpful. 8 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide lymphoid tissue.” MALT lymphoma can occur through- out the GI tract, but is common in the stomach, where it is almost invariably associated with underlying H. pylori gastritis. As such, gastric MALT lymphomas are derived from acquired MALT that develops in response to immunogenic bacteria, a process that is enhanced when the organism is a strain harboring the CagA gene. This lymphoma is composed predominantly of small-to-inter- mediate-sized B cells, many with a morphology recapitu- lating that of centrocytes in the normal lymphoid follicle. In addition, a variable proportion of the neoplastic cells have ample cytoplasm with indented nuclei, imparting a “monocytoid” appearance. Scattered large, “centroblast- like” cells are also common, but this should not be the predominant cell type as this appearance would be best classified as DLBCL. Some cases have plasmacytic dif- ferentiation and, occasionally, MALT lymphomas are FIGURE 4.4 BCL-2 negativity in a reactive germinal composed almost exclusively of plasma cells. Classically, center. Immunohistochemistry for BCL-2 staining can the lymphoma cells expand the mucosa and destroy the cause confusion in the workup of lymphoma in the GI normal mucosal structures, creating what are commonly tract and elsewhere. Many normal hematolymphoid cells, referred to as “lymphoepithelial lesions.” Macroscopically, including T cells, plasma cells, and mantle zone B cells, MALT lymphomas may cause expansion of the mucosal express BCL-2. In addition, many B cell lymphomas are folds, or produce ulcers. Unfortunately, this type of lym- BCL-2 positive. Thus, the best use of this immunostain is in the distinction between reactive, non-neoplastic germinal phoma has no specific immunophenotype, but the neo- centers like the one in the left of this photomicrograph, plastic B cells mark with CD20 and usually outnumber which are negative, and the neoplastic follicular structures associated T cells (unlike reactive lymphoid infiltrates, of follicular lymphoma, which are usually positive for BCL-2 in which T cells typically predominate). Around 30% to (see Chapter 9 for additional details and images). 50% of cases are reported to aberrantly coexpress CD43 on the neoplastic B cells, which can be a helpful feature when present. Finally, cases with plasmacytic differentia- tion contain abnormal plasma cells that will express only lymphoma is nodular, with neoplastic “follicles” that also kappa or lambda light chain (also known as light chain contain follicular dendritic cells, which can be highlighted restriction), a convenient marker of clonality when avail- with a CD21 or CD23 immunostain. FL is the one lym- able. The association of gastric MALT lymphoma with phoma where a BCL-2 immunostain can be crucial, as the H. pylori gastritis means that treatment of the underlying cells in the neoplastic follicles express this antigen in most inflammatory condition can actually cure the lymphoma cases of FL. This helps separate true FL from reactive fol- as well in most cases. Similarly, MALT lymphomas in licular hyperplasia (Figure 4.4). The common expression other sites respond to eradication/resolution of the inciting of BCL-2 by a very large number of other types of B cell inflammatory process, as typified by the mounting evi- lymphoma means that not everything that is BCL-2 posi- dence of an association between Campylobacter infection tive is FL, a point that commonly causes confusion. FL is and certain types of intestinal MALT lymphoma, which a relatively indolent lymphoma, but it commonly involves also respond to antibiotic therapy. the bone marrow and can be difficult to cure. A relatively recently described entity, dubbed “primary intestinal FL,” Follicular Lymphoma is usually found in the second part of the duodenum and is, by definition, isolated to the GI tract. This form of the The GI tract is often involved secondarily by follicular lym- disease, which has an identical immunophenotype to con- phoma that arises primarily in the retroperitoneal lymph ventional FL, is reported to be very indolent, and some nodes or elsewhere. Follicular lymphoma, as its name sug- patients have long-term survival even without treatment. gests, is a lymphoma of cells that recapitulate the follicle center B cells of a germinal center, and it is graded based on the proportion of small centrocyte-like cells (which often Mantle Cell Lymphoma have grooved or “cleaved” nuclei) to large centroblast-like Mantle cell lymphoma (MCL) has classically been associ- cells. Along with other B cell markers like CD20, the neo- ated with diffuse involvement of the GI tract that produces plastic B cells in FL express BCL-6 and CD10, which are the appearance of numerous polypoid projections of the markers of follicle center differentiation. The pattern of the mucosa that can mimic inherited polyposis syndromes. 4 General Approach to Lymphomas of the Gastrointestinal Tract 81 This so-called “lymphomatous polyposis” is regarded by B cell lymphoma and Burkitt lymphoma” in the most some as synonymous with GI involvement by MCL, but recent (2008) WHO classification. This diagnosis may this appearance has occasionally been reported in other have implications for response to therapy, as there is some types of lymphoma. MCL has a deceptively low-grade mor- evidence that such lymphomas may not respond well to phology, composed of small lymphocytes that often have conventional DLBCL-type chemotherapy. angulated nuclei, which can infiltrate diffusely or have a nodular pattern. Most confusing is the “mantle zone” T Cell Lymphomas pattern, in which the neoplastic mantle cells are arranged around reactive germinal centers, because this appearance While B cell lymphoma is by far the most common type can be easily overlooked as malignant and misdiagnosed of lymphoma involving the GI tract, there are two T cell as a reactive process. Despite its bland morphology, MCL lymphomas that merit mention in this brief introduction. is quite aggressive and has a median survival of only 3 to 5 First, enteropathy-associated T cell lymphoma (EATL) is, as years (although it is not believed to transform to DLBCL, the name suggests, usually related to an underlying gluten- unlike the other lymphomas described thus far). MCL has sensitive enteropathy (celiac disease). EATL may arise in a distinctive immunophenotype, aberrantly expressing the previously diagnosed celiac disease, or it may be the pre- pan-T cell marker CD5 as well as typical B cell markers senting event in undiagnosed patients, sometimes resulting like CD20. In addition, almost all cases express nuclear in perforation of the small intestine (often the jejunum) by a cyclin-D1, which usually confirms the diagnosis and dis- large, destructive lymphomatous mass. EATL may also be tinguishes MCL from GI involvement by chronic lympho- preceded by a phase of celiac disease that is refractory to a cytic leukemia/small lymphocytic lymphoma (CLL/SLL), gluten-free diet. As currently classified by the WHO, EATL another B cell lymphoma that expresses CD5. comes in two forms, one referred to simply as “EATL” and the other as “Type II EATL.” The Type II form comprises the minority of cases (up to 20%) and is composed of rather Burkitt Lymphoma monotonous small- and medium-sized lymphocytes that An aggressive B cell lymphoma, gastrointestinal Burkitt usually express CD8 and CD56, along with the pan-T cell lymphoma (BL) is classically associated with large masses marker CD3. This form is less often associated with under- involving the ileocecal region, but it can occur any- lying celiac disease. The cells in the “conventional” form of where in the GI tract. BL has several forms, including the the disease tend to be more pleomorphic and are most often endemic type, associated with Epstein–Barr virus (EBV) CD8 and CD56 negative, while also being CD3 positive. infection in certain parts of the world, and the sporadic The neoplastic T cells in this form also lose expression of type, found throughout the world. BL has an extraordi- CD5. EATL is an aggressive lymphoma, with poor overall narily high proliferative rate, with nearly 100% of cells survival. The association with an underlying malabsorption marking with the proliferation marker Ki-67 (Mib-1) in a syndrome likely contributes to the poor prognosis. typical case. This is also reflected in the easy identification Another T cell lymphoma that can involve the GI of mitotic figures. BL has a characteristic low-magnifica- tract, and which can be confused with EATL, is extra- tion appearance referred to as “starry sky,” because of the nodal NK/T cell lymphoma (ENKTL), nasal type. While numerous tingible-body macrophages admixed with the its name indicates the most frequent site of involvement, lymphoma cells. BL is composed of monotonous, medium- which is in and around the nose (where it was previously sized neoplastic cells with inconspicuous nucleoli, which termed “lethal midline granuloma,” among other names), mark with CD20, CD10, and BCL-6, although they are the GI tract is actually the most common extranasal site of almost always BCL-2 negative. Rearrangements in the involvement by this relatively rare entity. It tends to occur MYC gene are characteristic as well. BL is an aggressive in adults, and has a predilection for Asian patients and but potentially curable lymphoma, with an 80% to 90% native populations from Central America. Like EATL, survival rate when aggressively treated. In the past, lym- this is a clinically aggressive disease. It is composed of phomas that did not fit precisely the immunophenotype neoplastic T cells that express CD2 and CD56, but which and/or morphology of BL, but which had a similar high lack most other pan-T cell markers. The cells lack sur- proliferative rate, were termed “atypical BL,” although face CD3, but contain cytoplasmic CD3ε, which may be this terminology has recently fallen out of favor. Exactly identified by immunostains on paraffin-embedded tis- how to handle such lymphomas remains controversial, sue depending on the antibody specificity. In addition, but at least some have been found to harbor two or more the cells of ENKTL are essentially always positive for abnormalities or rearrangements in the MYC, BCL2, EBV by in situ hybridization (EBV-encoded ribonucleic and/or BCL6 genes, and have been dubbed “double-hit” acid [EBER] probe). They have a characteristic tendency lymphomas. Currently, these reside in the nebulous and to infiltrate the tissue with an angiocentric pattern that awkwardly named category of “B cell lymphoma, unclas- results in destruction of blood vessel walls, which also sets sifiable, with features intermediate between diffuse large this entity apart from EATL. 8 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide GENERAL APPROACH TO of CD43 is helpful in several ways. First, its expression LYMPHOMA DIAGNOSIS IN THE should normally roughly parallel that of CD3, as it is nor- GASTROINTESTINAL TRACT mally expressed on T cells. It is also

normally expressed by plasma cells, which are negative for CD20, so an infil- Approaches to evaluating GI specimens in which lym- trate heavy in these plasma cells (such as H. pylori gastri- phoma is being considered vary greatly, particularly when tis) may have increased CD43 staining. In addition, some dealing with small endoscopic tissue biopsies. While there B cell lymphomas aberrantly coexpress CD43, so definite is no absolutely correct or incorrect way to approach these staining in a B cell population is strong evidence for lym- diseases, a pragmatic diagnostic scheme that incorporates phoma. These CD43+ tumors include 30% to 50% of clinical, endoscopic, and morphologic features with ancil- MALT lymphomas, as well as those B cell neoplasms that lary testing (as needed) is outlined here. aberrantly coexpress CD5 (MCL and CLL/SLL). Finally, First, as mentioned earlier, the endoscopic impression expression of only CD43 by a hematolymphoid infiltrate of the lesion or process that was biopsied is crucial, and suggests that a myeloid neoplasm (such as GI involvement should be sought actively if it is not immediately available by acute myeloid leukemia) should be investigated. when evaluating the biopsy. The endoscopist’s viewpoint BCL-2 immunohistochemistry is often included in ini- serves as the pathologist’s “gross description” when deal- tial “lymphoma panels,” but this can create serious poten- ing with small biopsies, and this information is invaluable tial diagnostic pitfalls. There is a tendency to equate BCL-2 to both parties. For the pathologist, it provides additional expression with FL, but this is not the case. Expression of information regarding the extent and scope of the process, this antigen is common with a variety of normal lymphoid as a morphologically atypical lymphoid infiltrate takes on cells, including cells of the normal mantle zone surround- a more ominous tenor when associated with a large mass ing germinal centers, T cells, and plasma cells. In addition, or ulcer. For the practitioner performing the endoscopic most B cell lymphomas of any type express BCL-2. As sug- examination and clinical follow-up, the finding of a dis- gested earlier, the best use of this stain is to differentiate crete lesion provides a landmark that can be targeted dur- between reactive follicular hyperplasia and follicular lym- ing subsequent examinations, either to obtain more tissue phoma, and a diagnosis of the latter should be restricted to reach a definite diagnosis, or to monitor the progress of to a nodular/follicular pattern of B cells that express both potential therapy after the diagnosis is made. In addition, a marker of follicular center cells (CD10 and/or BCL-6) the pathologist must incorporate the background histo- and BCL-2 when it is used in this way. Additional evidence logic features found in the biopsy, including whether there for follicular structures can be obtained by using a marker is an underlying inflammatory condition (eg, H. pylori of follicular dendritic cells (CD21 and/or CD23). infection) that may be contributing to the development The use of additional ancillary studies when evalu- of lymphoma, or evidence of underlying gluten-sensitive ating putative GI lymphomas, particularly molecular enteropathy in the case of EATL. diagnostic assays and flow cytometry, is somewhat con- Once the pathologist determines that there is suffi- troversial. The tissue typically obtained by endoscopic cient suspicion of lymphoma to warrant further diagnostic biopsy is scant enough that it is often most prudent to workup, he or she typically selects a battery of immunos- conserve it for a good look at the hematoxylin and eosin tains. While it can be tempting to try to be comprehensive (H&E)-stained morphology combined with judicious and all-encompassing on the first pass, a tailored approach immunohistochemistry, rather than attempting to subdi- that takes into account the entities with the highest “pre- vide it and send precious tissue (which may or may not test probability” is favored, as there is a significant risk of contain tumor) to other labs. This is particularly true for too much information when large immunostain panels are flow cytometry, which is suboptimal for diagnosis of the used at the outset. In addition, one does not want to cut most common type of GI lymphoma (DLBCL) due to poor entirely through the tissue in a small biopsy block when cell survival during the disaggregation processes used to performing a large battery of immunostains, only to find prepare tissue for flow cytometric analysis. Evaluation of out that different stains are needed, yet there is no tissue GI biopsies by flow cytometry can also be affected by con- left in the block. tamination by epithelial cells. There are a few situations Specific staining patterns and approaches will be dis- where molecular studies can be quite helpful, but often cussed in Chapters 8, 9, and 11, but a reasonable first-tier after the actual diagnosis of lymphoma is made. Such sit- set of stains for most cases is CD3, CD20, and CD43. uations will be addressed in Chapters 8, 9, and 11, but This panel allows the “balance” of T and B cells to be two worth mentioning are H. pylori-related MALT lym- ascertained, and a predominance of small T cells with phoma and aggressive large B cell lymphomas where the fewer B cells often points toward a reactive phenomenon. differential diagnosis includes DLBCL with a high pro- In addition, the architecture of the infiltrate can often liferative rate and the so-called “double-hit” B cell lym- be better seen when highlighted with immunostains, as phoma. Some cases of H. pylori-related MALT lymphoma B cell follicles are more easily recognizable. The addition harbor a specific translocation, t(11;18)(q21;q21), that is 4 General Approach to Lymphomas of the Gastrointestinal Tract 8 3 associated with poor or absent response to conservative small polyps. Similar tissue can be seen in the right therapy aimed at H. pylori eradication, so identification colon, and the normally abundant lymphoid tissue sur- of this abnormality by molecular diagnosis may be helpful rounding the appendiceal orifice can prompt a biopsy as in avoiding a fruitless trial of conservative therapy. Some well. Under the microscope, even normal lymphoid tissue authors recommend performing this test immediately at in an expected location can have a worrisome appear- the time of initial diagnosis, even though the vast majority ance, depending on the plane of section, the interaction (greater than 80%–90%) of gastric and duodenal MALT with the associated mucosal structures, and the archi- lymphomas will respond to antibiotic therapy (even when tecture. Worrisome microscopic findings combined with H. pylori is not identified in biopsies). As discussed earlier, certain clinical settings, such as a middle-aged or older “double-hit” B cell lymphomas harbor molecular abnor- patient in whom prominent lymphoid tissue is somewhat malities in two or all three of the genes MYC, BCL2, surprising, can result in an expensive diagnostic workup and BCL6, and this information can help to cement the that is undertaken in a valiant effort to avoid missing a diagnosis and differentiate these aggressive lymphomas diagnosis. from conventional DLBCL, as well as potentially affect- In this situation, it is important to understand what ing therapy. can create an “atypical” appearance in the normal lym- As with flow cytometric analysis and other molecu- phoid population of the right lower quadrant, and to com- lar studies, the question of whether or not to order B or bine this with an understanding of just how prominent the T cell gene rearrangement testing is one that can create lymphoid aggregates in this region can be. A few specific struggles when making decisions about tissue allocation, features are most often the cause of diagnostic problems. especially for small diagnostic biopsies. This is further The first is the interaction of lymphoid tissue with the complicated by the fact that reactive, non-neoplastic T fibers of the muscularis mucosae. Some observers regard cell populations and, to an extent, B cell populations as any admixture of lymphocytes with the muscularis muco- well, can harbor small clonal populations. When sub- sae as evidence of aggressive or “invasive” behavior. In jected to amplification, these can potentially lead to an reality, however, odd planes of section, mild mucosal pro- overdiagnosis of lymphoma. Therefore, while gene rear- lapse changes, and other normal variants can cause groups rangement studies can sometimes “tip the balance” and of lymphocytes to seemingly mix with the smooth muscle provide evidence for neoplasm in difficult or equivocal fibers; thus this is not necessarily evidence of malignancy. cases, it may be a better use of precious tissue to ensure Large lymphoid aggregates can also give an impression a good look at H&E morphology and carefully selected of mucosal “distortion,” when normal crypts are pushed immunohistochemical results in making a diagnosis of out of the way and villi seem shortened, and individual lymphoma, particularly because accurate subclassifica- lymphocytes can infiltrate the epithelium of the adjacent tion is often important. crypts, mimicking lymphoepithelial lesions of the type seen especially in MALT lymphomas (Figure 4.5). Finally, and perhaps most confusing, is the primary follicle. While SPECIFIC ILLUSTRATIVE EXAMPLES we expect to encounter germinal centers in Peyer’s patches and other large lymphoid aggregates such as those associ- To help illustrate the concepts outlined in this introduc- ated with the appendix, a tangential section or a sample tory chapter on a pragmatic approach to the diagnosis of containing primary follicles that have not developed into GI lymphomas, two specific clinical scenarios are worth germinal centers can present the pathologist with nod- exploring in more depth. ules of monotonous mantle cells, which normally express BCL-2 (Figure 4.6). This is another important scenario Lymphoid Tissue in the Terminal Ileum where BCL-2 positivity is not equivalent to FL. and Right Colon A common question often asked in consultation is, “How Gastritis Versus MALT Lymphoma much lymphoid tissue is normal in the terminal ileum and A second source of many specialty consultations is the ques- right colon?” The somewhat tongue-in-cheek answer is, tion of gastric MALT lymphoma, particularly the effort to “It depends.” understand exactly where severe H. pylori gastritis ends Prominent lymphoid tissue is the norm in the right and early MALT lymphoma begins. Unfortunately, there lower quadrant, particularly in young patients, but not is no easy answer to this question, but application of a uncommonly in patients of any age. Peyer’s patches in the pragmatic approach can ease the pain of trying to provide terminal ileum, a form of “native MALT,” can be quite one. Several features of this low-grade lymphoma make prominent, and as mentioned in the Unique Features of it potentially challenging. First, it is quite common and it Gastrointestinal Lymphoid Tissue section, may impart is usually relatively easily treated, so the index of suspi- an endoscopic picture of mucosal “nodularity” or even cion tends to be high when any lymphoid tissue is found in 8 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 4.5 Effects of a benign Peyer’s patch on FIGURE 4.6 This ileal biopsy contains a lymphoid mucosal structures. In addition to displacing the crypts and aggregate without an organized germinal center (Peyer’s causing a localized blunting of overlying villi as illustrated patch with primary lymphoid follicle). Otherwise, it has in Figure 4.1, the lymphocytes of a normal Peyer’s patch similar features to the Peyer’s patch seen in Figure 4.1, with can both interdigitate with the fibers of the muscularis mild architectural disorganization and small lymphocytes mucosae, seen at the bottom of this image, and infiltrate infiltrating the adjacent surface and crypt epithelium. the epithelium of surrounding crypts. Neither of these Characterizing this lymphoid population with immunostains features is diagnostic of a neoplastic process. When would reveal a mixture of T cells and B cells, but the lymphocytes are found within the epithelium in a benign latter would also likely express BCL-2, because many of condition, the destructive infiltration characteristic of the the B cells are mantle zone lymphocytes. This can cause “lymphoepithelial lesions” seen in MALT lymphoma is confusion with lymphoid neoplasms, particularly follicular absent (see Chapter 8 for additional details and images). lymphoma (see also Chapter 9). the gastric mucosa. The GI tract is the most common site the suspicion of MALT lymphoma are commonly found for MALT lymphoma, and 85% of GI cases occur in the in biopsies that come from mucosa with rather nebulous stomach in association with H. pylori gastritis. In addi- endoscopic findings such as “gastritis” or “erythema.” tion, there are no specific

immunohistochemical markers In this setting, one must be careful with an unequivocal for MALT lymphoma, so the diagnosis relies heavily on diagnosis of lymphoma. It can be impossible, without a the H&E morphology coupled with the balance of T and B targetable endoscopic finding, for the patient to be easily lymphocytes identified by appropriate immunostains (and, monitored following diagnosis. Thus, in cases where the on occasion, B cell gene rearrangement studies). Extensive histologic suspicion is very high and the sample is consid- plasmacytic differentiation can further confuse the issue, ered too scant for a definite diagnosis, it may be best for because a dense infiltrate of plasma cells can be regarded the patient to undergo another examination that can bet- simply as part of the underlying gastritis and overlooked ter “map” the mucosa and obtain more tissue for diagno- as a part of a neoplasm. Evidence of clonality using kappa sis. When the suspicion is relatively low, the very high rate and lambda light chain immunohistochemistry can easily of MALT lymphoma response to conservative H. pylori solve this problem, and this should be kept in mind when eradication therapy means that treatment of the underly- evaluating a lymphoid infiltrate with plasmacytic features. ing disease is very likely to be sufficient in eradicating a Even when extensive plasmacytic differentiation is consid- possible lymphoma, in any case, and prudence may dictate ered, there is another potential stumbling block; the use simply treating the gastritis rather than wasting time and of a CD138 stain to highlight the plasma cells can lead to energy on wrestling with the diagnosis. a different type of misdiagnosis, as many epithelial and Even when accompanied by a finding of diffusely mesenchymal neoplasms also express CD138. thickened gastric folds or a malignant-appearing ulcer, the As noted earlier, correlation with the endoscopic diagnosis of MALT lymphoma, while easier to make, is impression is important in the diagnosis of any lymphoma not without potential pitfalls. In particular, earlier-than- in the GI tract, but this is particularly true with regard optimal rebiopsy to assess response to therapy can do to MALT lymphoma. Because of the overlap with severe more harm than good, and can lead to confusion. While H. pylori gastritis, atypical lymphoid infiltrates that raise most MALT lymphomas are sensitive to eradication of the 4 General Approach to Lymphomas of the Gastrointestinal Tract 8 5 inciting H. pylori organisms, this process can take months Sagaert X, Tousseyn T, Yantiss RK. Gastrointestinal B-cell lymphomas: to reach full histologic resolution. Cases taking greater from understanding B-cell physiology to classification and molecu- lar pathology. World J Gastrointest Oncol. 2012;4:238–249. than a year to resolve have even been reported. Thus, rebi- Smith LB, Owens SR. Gastrointestinal lymphomas: entities and mimics. opsy at 4 or 6 weeks may result in an appearance that Arch Pathol Lab Med. 2012;136:865–870. is not appreciably different from the original diagnostic biopsy (although the success of H. pylori eradication may be assessed). Treatment of MALT lymphomas considered B Cell Lymphomas refractory to conservative therapy may be escalated to che- Harris NL, Swerdlow SH, Jaffe ES, et al. Follicular lymphoma. motherapy or other modalities, so it is important not to In: Swerdlow SH, Campo E, Harris NL, et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid give the impression of refractoriness too early in the course Tissues. Lyon, France: IARC Press; 2008:265–266. of treatment. It is reasonable to diagnose “residual MALT Leoncini L, Raphael M, Stein H, et al. Burkitt lymphoma. In: Swerdlow lymphoma” and to comment on how the infiltrate com- SH, Campo E, Harris NL, et al., eds. WHO Classification of pares to earlier biopsies (eg, “significantly improved from Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008:262–264. prior biopsy on [DATE]”) to provide a record of continu- Owens SR. Large cell lymphoma. In: Greenson JK, Lamps LW, ing progress in resolution of the lymphoma. Montgomery EA, et al. Diagnostic Pathology: Gastrointestinal. Finally, the frequency of gastric MALT lymphoma Salt Lake City, UT: Amirsys, Inc. 2010:2.76–2.79. may induce a tendency to regard almost any lymphoma Owens SR. MALT lymphoma. In: Greenson JK, Lamps LW, Montgomery EA, et al. Diagnostic Pathology: Gastrointestinal. in the stomach as this type of disease. It is important to Salt Lake City, UT: Amirsys, Inc. 2010:2.70–2.75. consider the possibility of other types of lymphoma, in Owens SR. Mantle cell lymphoma. In: Greenson JK, Lamps LW, particular DLBCL, because these may not respond to Montgomery EA, et al. Diagnostic Pathology: Gastrointestinal. H. pylori eradication therapy. Furthermore, DLBCL can Salt Lake City, UT: Amirsys, Inc. 2010:2.80–2.83. Psyrri A, Papageorgiou S, Economopoulos T. Primary extranodal lym- evolve from MALT lymphoma, so recognition of a large phomas of stomach: clinical presentation, diagnostic pitfalls and cell component is very important, and will be discussed in management. Ann Oncol. 2008;19:1992–1999. greater detail in Chapter 8. Zullo A, Hassan C, Ridola L, et al. Gastric MALT lymphoma: old and new insights. Ann Gastroenterol. 2014;27:27–33. SELECTED REFERENCES T Cell Lymphomas General Chan JKC, Quintanilla-Martinez L, Ferry JA, Peh S-C. Extranodal NK/T-cell lymphoma, nasal type. In: Swerdlow SH, Campo Burke JS. Lymphoproliferative disorders of the gastrointestinal tract: A E, Harris NL, et al., eds. WHO Classification of tumours of review and pragmatic guide to diagnosis. Arch Pathol Lab Med. Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2011;135:1283–1297. 2008:285–288. Dickson BC, Serra S, Chetty R. Primary gastrointestinal tract lym- Ferreri AJ, Zinzani PL, Govi S, Pileri SA. Enteropathy-associated T-cell phoma: diagnosis and management of common neoplasms. Expert lymphoma. Crit Rev Oncol Hematol. 2011;79:84–90. Rev Anticancer Ther. 2008;6:1609–1628. Isaacson PG, Chott A, Ott G, Stein H. Enteropathy-associated T-cell O’Malley DP, Goldstein NS, Banks PM. The recognition and classifi- lymphoma. In: Swerdlow SH, Campo E, Harris NL, et al., cation of lymphoproliferative disorders of the gut. Hum Pathol. eds. WHO Classification of Tumours of Haematopoietic and 2014;45:899–916. Lymphoid Tissues. Lyon, France: IARC Press; 2008:289–291. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract LAUR A W. LAMPS AND MAT THEW R . LINDBERG INTRODUCTION GENERAL APPROACH TO SPECIMEN EVALUATION AND DIAGNOSIS Despite their overall rarity when compared to epithelial and other nonmesenchymal tumors, mesenchymal neo- Clinical Evaluation plasms are relatively common in the gastrointestinal (GI) tract. This large group of tumors encompasses a wide range The importance of the location of a mesenchymal tumor of entities, including both conventional soft tissue enti- within the GI tract cannot be overemphasized. Some mes- ties (eg, lipoma, leiomyoma, and nerve sheath tumors) as enchymal tumors show a predilection for particular sites well as entities that are almost entirely restricted to the GI and are very uncommon in others. For example, the vast tract (eg, gastrointestinal stromal tumor [GIST], plexiform majority of mesenchymal neoplasms in the stomach are fibromyxoma, Schwann cell hamartoma, and inflamma- GISTs, whereas in the esophagus mesenchymal tumors are tory fibroid polyp). GIST has the distinction of being the usually leiomyomas. Not uncommonly, a reasonable guess most common GI mesenchymal tumor overall, c omprising can be made as to the diagnosis before a histologic section 0.2% of all GI tumors. is even seen, just by knowing the anatomic site. This infor- Many pathologists are uncomfortable with the diagno- mation is summarized in Table 5.1. sis of mesenchymal tumors, but GI mesenchymal neoplasms Knowledge of the patient’s clinical history may also tend to pose less of a problem than their counterparts arising provide useful information. For example, a solitary mural from soft tissue due to a more limited differential diagnosis. tumor arising in a patient with neurofibromatosis is usu- Histomorphologic evaluation, accompanied by basic clini- ally a GIST (and not a malignant peripheral nerve sheath cal information (eg, site) and a small panel of immunohisto- tumor), whereas multiple serosal or transmural nodules in chemical stains, is often sufficient to accurately classify a GI the same demographic likely represent plexiform neuro- mesenchymal neoplasm. This chapter will discuss the basic fibromas. In addition, the history of another malignancy approach to the evaluation of mesenchymal neoplasms, fea- (such as primary lung carcinoma or malignant melanoma) tures of GISTs and their differential diagnosis, and current should always lead the pathologist to exclude a metastasis ancillary testing that is increasingly considered the standard before investigating the possibility of a primary mesenchy- of care from both a prognostic and a therapeutic standpoint. mal neoplasm. 87 8 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 5.1 Distribution of Mesenchymal Tumors by Site in the Gastrointestinal Tract Esophagus Stomach Small Bowel Colon Anus Leiomyoma GIST GIST GIST (particularly Leiomyoma rectum) Granular cell tumor Schwannoma Inflammatory fibroid polyp Leiomyoma GIST (particularly ileum) Giant fibrovascular Inflammatory fibroid Granular cell tumor Granular cell tumor Granular cell polyp polyp (antrum) (duodenum) tumor GIST Glomus tumor Perineurioma Ganglioneuroma Schwannoma Gross Evaluation hematoxylin and eosin (H&E) sections have been evalu- ated and any necessary ancillary diagnostic techniques When approaching a gross specimen for evaluation of have been incorporated. The differential diagnosis of epi- a mesenchymal neoplasm, it is particularly important thelioid tumors on frozen section is, of course, more broad to document the size of the tumor (this is particularly and challenging. If there is any chance that that tumor important for GISTs, as size is an important prognos- is an epithelioid mesenchymal neoplasm (mainly GIST), tic indicator). If possible, the specific anatomic layer (eg, a preliminary diagnosis of “epithelioid neoplasm” with submucosa, muscularis propria) of origin within the wall deferral to evaluation of permanent sections is warranted. of the GI tract should be noted as well (see Table 5.2). If the tumor appears to be extraintestinal, such as from the mesentery or omentum, then its relationship to the Histologic Evaluation GI tract and/or other organs in the resection specimen A reasonable histologic approach to GI tract mesenchymal should be described. The presence of necrosis should tumors includes the following: be noted. Margins should be sampled, if applicable, and multiple sections of the tumor submitted (ideally • Determination of growth pattern (circumscribed, one section per centimeter of the greatest tumor dimen- infiltrative, polypoid, etc.). sion). Consideration should also be given to reserving • Evaluation of overall cellular morphology (spindled, fresh tissue for molecular analysis or cytogenetic studies, epithelioid, or mixed). although the majority of ancillary tests can be performed • Assessment of the presence or absence of nuclear directly from the paraffin block. atypia/pleomorphism. • Assessment for characteristic features that may be of Intraoperative Evaluation diagnostic utility. Frozen section evaluation may be requested if the tumor Growth Pattern type is in question at the time of surgery. For many mes- enchymal tumors, the intraoperative diagnosis of “spindle The differential diagnosis of a discrete mesenchymal polyp cell neoplasm” is sufficient, with the caveat that a formal is often fairly limited and well-defined, whereas a submuco- diagnosis must be deferred until multiple formalin-fixed sal or mural mass engenders a broader list of considerations, TABLE 5.2 Classification of GI Mesenchymal Tumors by Location in the Wall of the GI Tract Mucosa Submucosa Muscularis Propria Serosa/Mesentery Nerve sheath/neural tumors Inflammatory fibroid polyp GIST Desmoid tumor Perineurioma Nerve sheath tumors Schwannoma Inflammatory myofibroblastic tumor Leiomyoma of muscularis Gangliocytic paraganglioma Leiomyoma Sclerosing mesenteritis mucosae Kaposi’s sarcoma Lipoma Glomus tumor Extraintestinal GIST Source: Adapted from Voltaggio L. and Montgomery EA: “Gastrointestinal spindle cell lesions: just like real estate, it’s all about location.” Mod Pathol. 2015;28 Suppl 1: S47-66. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 8 9 including rarer entities. Some of the more common mes- Nuclear Atypia/Pleomorphism enchymal polyps include ganglioneuroma, leiomyoma, Significant nuclear atypia/pleomorphism is generally perineurioma, Schwann cell hamartoma, inflammatory uncommon in GI tract mesenchymal neoplasms. When fibroid polyp, and granular cell tumor, all of which are faced with a pleomorphic submucosal or mural neoplasm, benign and generally of little clinical consequence. Entities metastatic carcinoma and melanoma should always be such as GIST, schwannoma, mural leiomyoma/leiomyo- promptly excluded first. Once this is done, leiomyosar- sarcoma, plexiform angiomyxoma, and glomus tumor are coma should be considered next, followed by entities such more likely to present clinically as masses, and not polyps. as GIST, symplastic leiomyoma, and other rare tumors. Importantly, as noted earlier, when dealing with a nonpol- As a rule, nuclear pleomorphism in GIST is very uncom- ypoid subepithelial tumor, the anatomic layer of origin is mon, even in malignant cases. Two notable exceptions to important to note and

often provides a useful clue to the this rule are pleomorphic epithelioid GIST, which may diagnosis (see Table 5.2). For example, GIST arises in the show prominent nuclear atypia (resembling carcinoma muscularis propria, while schwannoma and inflammatory or melanoma) but almost no mitotic figures (Figure 5.1); fibroid polyp arise in the submucosa. Inflammatory myofi- and dedifferentiated GIST, which is defined as a classic broblastic tumor (IMFT) and desmoid tumor usually arise KIT-positive spindled GIST containing an anaplastic KIT- extrinsic to the GI tract (ie, mesentery) but may appear to negative component (Figure 5.2A–B). arise in the serosa or the muscularis propria (if involved through local extension). Characteristic/Diagnostic Features Overall Cellular Morphology There are a few histologic findings or patterns that, if The differential diagnosis of a spindled mesenchymal neo- present, can help significantly narrow a broad differential plasm is quite broad, but most commonly includes GIST, diagnosis. Nuclear palisading in a GI tract mesenchymal schwannoma, leiomyoma/leiomyosarcoma, and inflam- tumor often indicates a GIST (Figure 5.3), despite its tradi- matory fibroid polyp. Knowing what portion of the GI tional association with neural neoplasms such as schwan- tract is involved, as noted previously in the section on gen- noma. Peritumoral or intratumoral lymphoid aggregates eral approach to specimen evaluation and in Table 5.1, is (Figure 5.4) should suggest schwannoma, although they often very helpful in narrowing down this list. Epithelioid can be seen in other tumors as well. A multinodular or neoplasms are generally more straightforward and almost plexiform growth pattern suggests plexiform fibromyx- always represent GISTs, provided that carcinoma and mel- oma, plexiform neurofibroma, or succinate dehydro- anoma are fully excluded. Glomus tumor is also a note- genase (SDH)-deficient GIST (Figure 5.5). Prominent worthy consideration. Tumors with a mixture of spindled myxoid stroma is a feature of many GISTs, but can also and epithelioid cells are also usually GISTs. This informa- be seen in smooth muscle tumors, plexiform fibromyx- tion is summarized in Table 5.3. oma, inflammatory fibroid polyp, IMFT, and desmoid TABLE 5.3 Approach to Gastrointestinal Mesenchymal Neoplasms Based on Histologic Pattern Small Round Spindle Cell Epithelioid Nested Myxoid Blue Cells Pleomorphic GIST GIST Gangliocytic GIST DSRBCT Leiomyosarcoma Paraganglioma Desmoid PEComa Clear-cell-sarcoma-like Inflammatory fibroid Round cell MPNST tumor of the GI tract polyp liposarcoma Schwannoma Schwannoma Plexiform fibromyxoma GIST Leiomyoma/ Epithelioid vascular Leiomyosarcoma Liposarcoma leiomyosarcoma tumors Inflammatory fibroid Glomus IMFT polyp Perineurioma Granular cell tumor Neurofibroma Neurofibroma Rhabdoid tumor Inflammatory pseudotumors IMFT SFT Plexiform fibromyxoma Granular cell tumor Abbreviations: DSRBCT, desmoplastic small round blue cell tumor; IMFT, inflammatory myofibroblastic tumor; MPNST, malignant peripheral nerve sheath tumor; PEComa, perivascular epithelioid cell tumor; SFT, solitary fibrous tumor. 9 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide larger tumor, and therefore may not be representative, leading to misdiagnosis or undergrading. This is particu- larly problematic with GIST, as modern prognostication schemes require careful evaluation of numerous micro- scopic fields for the presence of mitotic figures. Accurate grading is often impossible on a small biopsy, particularly if only a few or no mitoses are present. Radiographic cor- relation may be helpful when evaluating needle biopsies of mesenchymal neoplasms, as they can provide informa- tion on size, relationship to anatomical structures, and the presence or absence of possible metastases. Immunohistochemical Evaluation Immunohistochemistry can be extremely helpful in the evaluation of GI tract mesenchymal tumors. The most FIGURE 5.1 Pleomorphic GIST may show striking commonly utilized antibodies include KIT, DOG1, S100 nuclear atypia, as seen here in a gastric tumor, but mitotic protein, smooth muscle actin (SMA), desmin, CD34, and figures are scarce instead of not increased. broad-spectrum keratin. A summary of the basic immu- nohistochemical features and pitfalls in the evaluation of GI mesenchymal tumors is given in Table 5.4. It is extremely important to be aware of the sensitivity, speci- tumor. Large dilated/ectatic stromal vessels (ie, “stag- ficity, and potential cross-reactivity of immunostains that horn” or hemangiopericytoma-like) may be seen in GIST, are used in diagnosis and evaluation of mesenchymal though not nearly as often or as prominently as in solitary tumors of the GI tract (or any other tumor), as well as fibrous tumor, which of course must be carefully excluded staining artifacts. (Figure 5.6A–B). Tumor involvement of the mucosa is not typical of malignant GIST, and should lead to consider- KIT and DOG1 ation of leiomyosarcoma, metastatic carcinoma/mela- noma, and other malignancies (Figure 5.7). Diffuse KIT expression is characteristic of GIST Care must be taken when evaluating a GI tract mesen- (Figure 5.8A), and may be cytoplasmic or membranous chymal tumor that has been sampled only by a small biopsy. (but not nuclear). It rarely shows a perinuclear dot-like pat- Such biopsies are often only a minute portion of a much tern of expression (Figure 5.8B). DOG1 is nearly identical (A) (B) FIGURE 5.2 Dedifferentiated GIST is defined as a typical spindle cell GIST with characteristic immunostaining, which also contains an anaplastic component (A) that is either KIT negative or only weakly positive (B). Figures courtesy of Dr. Jason L. Hornick. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 91 FIGURE 5.5 A multinodular or plexiform growth pattern, as seen here in a GIST, suggests either SDH-deficient FIGURE 5.3 Despite the traditional association with GIST or plexiform fibromyxoma. schwannomas, GISTs frequently feature areas of nuclear palisading. in sensitivity and specificity to KIT and may be used as an alternative. It may also be used in conjunction with KIT to increase sensitivity. Rare GISTs are KIT negative (usually those with epithelioid morphology), but DOG1 is often positive in these tumors. Exceptionally rare tumors are both KIT and DOG1 negative, and often require molecular analysis to make a diagnosis. (A) (B) FIGURE 5.6 Although dilated or ectatic vessels (also known as “staghorn” or “hemangiopericytoma-like” FIGURE 5.4 Peritumoral and/or intratumoral lymphoid vessels) may be seen in GIST (A), they are more often aggregates are very common in gastrointestinal tract associated with solitary fibrous tumor (B). However, schwannomas, as seen here in this gastric tumor. solitary fibrous tumor of the GI tract is very rare. 9 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Caveats KIT is expressed by mast cells; thus it is important to dif- ferentiate KIT-positive mast cells from true tumor cells when intratumoral mast cells are prominent within a non- GIST mesenchymal tumor (Figure 5.9). One should always be reluctant to diagnose a GIST in the setting of scattered KIT-positive individual cells. KIT expression has been rarely reported in desmoid tumors, and DOG1 expression has been rarely reported in leiomyosarcomas and malignant periph- eral nerve sheath tumors. KIT also stains a wide variety of nonmesenchymal tumors, including melanoma, Kaposi’s sarcoma, plasma cell myeloma, urothelial carcinoma, chro- mophobe renal cell carcinoma, thymic carcinoma, and some breast carcinomas (Figure 5.10A–B). DOG1 positivity can also be seen in synovial sarcoma, gastric adenocarcinoma, acinic cell carcinoma of salivary gland, and rarely in malig- nant peripheral nerve sheath tumors. S100 PROTEIN FIGURE 5.7 True mucosal invasion is not typical of GIST, Strong, diffuse nuclear and cytoplasmic expression is and should lead to consideration of other entities such as characteristic of schwannoma, Schwann cell hamartoma, malignant melanoma, as shown here. granular cell tumor, and ganglioneuroma (Figure 5.11). TABLE 5.4 Basic Panel of Immunostains Useful in the Diagnosis of Mesenchymal Tumors of the GI Tract GIST Leiomyoma Leiomyosarcoma Schwannoma Desmoid KIT 95% Negative Negative Negative Rarely positive DOG1 ~90% Negative 0.3% Negative Negative CD34 70% Negative Negative Negative Negative SMA 30%–40% ~100% 86% Negative Negative Desmin 1%–2% ~100% 50%–80% Negative Negative S100 1%–2% Negative Negative 100% Negative Cytokeratin 1%–2% ~20% (varies with keratin used) 20%–38% Rare, focal Negative (A) (B) FIGURE 5.8 Diffuse cytoplasmic or membranous KIT expression is characteristic of GIST (A), but staining should not be nuclear. Rarely, KIT immunostaining produces a perinuclear dot-like pattern of expression (B). 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 9 3 FIGURE 5.9 This KIT stain of a duodenal leiomyoma shows scattered intratumoral mast cells. KIT-positive mast cells should not be mistaken for staining of tumor cells. FIGURE 5.11 This gastric Schwann cell hamartoma shows strong, diffuse staining for S100. Caveats Scattered individual S100-positive cells within a tumor are likely dendritic-type cells. A proportion of GISTs that arise within the setting of neurofibromatosis also express S100 protein. Smooth Muscle Actin Diffuse cytoplasmic expression (Figure 5.12A) is most characteristic of smooth muscle neoplasms such as leio- myoma and leiomyosarcoma, but SMA positivity is also present in glomus tumors and PEComas. Expression can also be seen in plexiform fibromyxoma, IMFT, and des- (A) moid, but it is usually more “wispy” and less impressive in these tumors (Figure 5.12B). Caveats In approximately 20% of cases, GIST can show focal to extensive SMA positivity (Figure 5.13). It may also be focally expressed in some gastric schwannomas. Desmin Strong, diffuse desmin expression is typical of smooth muscle neoplasms, particularly leiomyoma (Figure 5.14). Leiomyosarcoma is negative in approximately half of the cases. PEComa, desmoid, and IMFTs may also show patchy expression. (B) Caveats FIGURE 5.10 This epithelioid thymic carcinoma (A) shows Desmin may be expressed in rare cases of gastric schwan- strong, diffuse cytoplasmic and membranous staining for noma. In the GI tract, focal expression of desmin in the KIT (B). Figures courtesy of Dr. Jason L. Hornick. absence of SMA or similar antibodies is likely nonspecific. 9 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 5.12 Diffuse cytoplasmic expression of smooth muscle actin is most characteristic of smooth muscle neoplasms, as seen here in a gastric leiomyoma of the muscularis mucosae (A). Wispy, weaker SMA expression can be seen in a variety of other tumors, including inflammatory myofibroblastic tumor (B, courtesy of Dr. Andrew Folpe). CD34 Keratin CD34 expression is seen in a variety of tumors including In the evaluation of GI mesenchymal tumors, keratin GIST, solitary fibrous tumor, and inflammatory fibroid is primarily used to exclude carcinoma. Diffuse cyto- polyp, and therefore it is of limited utility in evaluating plasmic expression is typical of epithelial malignancies. mesenchymal tumors of the GI tract. Focal expression is nonspecific, but may also indicate carcinoma. Caveats Many mesenchymal tumors, both intrinsic and extrinsic Caveats to the GI tract, express CD34. If it is used at all, it should Keratin may be focally expressed in gastric schwannomas. be used as part of a panel rather than as a confirmatory Some antibodies (such as cytokeratin [CK] AE1/AE3) may stain on its own. show patchy expression in smooth muscle tumors as well. FIGURE 5.13 This needle biopsy of a small bowel GIST FIGURE 5.14 Strong, diffuse desmin expression is shows strong SMA positivity. typical of smooth muscle tumors, particularly leiomyomas. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 9 5 Other Markers molecular characteristics. In addition, molecular and cytogenetic testing has become the standard of care in the Nuclear β-catenin expression may be helpful in supporting diagnosis, prognosis, and therapeutic decision making in the diagnosis of desmoid tumor (Figure 5.15). Melanocytic many GI mesenchymal tumors. A summary of molecular markers (HMB-45 and MART-1) are used mainly to eval- alterations and correlative tests is given in Table 5.5. uate for metastatic melanoma, but these markers are often coexpressed with SMA in PEComa (Figure 5.16). Focal MART-1 expression has also been reported in 30% to Diagnostic Pitfalls 40% of epithelioid GISTs. The majority of diagnostic errors that occur when evaluat- ing mesenchymal tumors of the GI tract are due to either Molecular Evaluation failure to consider spindle cell lesions other than GIST in the differential diagnosis, or to incorrect interpretation of Some mesenchymal tumors of the GI tract, particu- immunostains and lack of awareness of the spectrum of larly GISTs but not exclusively so, have well-elucidated tumors that commonly used immunostains can mark (see section on immunohistochemical evaluation). COMMON MESENCHYMAL TUMORS OF THE GI TRACT Gastrointestinal Stromal Tumors GISTs are the most common mesenchymal tumor of the GI tract. GISTs are believed to arise from the KIT-positive interstitial cells of Cajal, which are an innervated net- work of cells associated with Auerbach’s plexus. These are known as “intestinal pacemaker cells,” as they coordinate peristalsis. This chapter will provide a general overview and approach to these tumors, but additional discussion may be found in Chapters 8, 9, and 11. GISTs present within a wide patient age range, with a peak in the fifth to seventh decades and an equal

gen- FIGURE 5.15 Nuclear β-catenin expression is helpful in der distribution. Symptoms depend on location and size supporting the diagnosis of desmoid tumors. Cytoplasmic of the tumor, and a significant proportion are incidental expression is entirely nonspecific and is not diagnostically useful. findings in imaging studies or procedures for unrelated conditions. The most common symptoms are nonspecific upper abdominal pain and GI bleeding. Other presenting signs and symptoms include bloating, early satiety, signs/ symptoms of a mass, or obstruction in small bowel GIST. Pathologic Features GISTs are most common in the stomach (60% of tumors) followed by the small bowel (30%), esophagus, and col- orectum (approximately 5% each). One percent or less of GIST arise in the omentum and mesentery, and are known as extraintestinal GIST (EGIST). Grossly, GISTs arise in the muscularis propria, and are usually somewhat centered in the wall of the tubular gut (Figure 5.17A–C). They vary widely in size, ranging from 1.0 mm to more than 40 cm, with a median size of 6 cm in the stomach, 4.5 cm in the duodenum, and 7.0 cm in the distal small bowel. Tumors may occasionally protrude into the lumen in a polypoid fashion (Figure 5.17D), and show overly- FIGURE 5.16 PEComas express HMB-45 (shown here, ing mucosal ulceration. The cut surface may show hem- courtesy of Dr. Andrew Folpe) as well as SMA. orrhage, calcification, or, less commonly, necrosis. GISTs 9 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 5.5 Summary of Molecular Alternations in GI Mesenchymal Tumors GIST IFP IMFT Desmoid CSS Affected gene KIT PDGFRA (55%–70%) ALK APC (FAP cases) EWSR1 PDGFRA CTNNB1 (most sporadic SDH cases) Test KIT IHC; PDGFRA mutational testing IHC for ALK protein (results IHC for nuclear β-catenin RT-PCR; SDH IHC; (usually not necessary in very variable) FISH Mutational analysis this context) FISH Abbreviations: CSS, clear cell sarcoma; IFP, inflammatory fibroid polyp; IMFT, inflammatory myofibroblastic tumor; FAP, familial adenomatous polyposis. are typically circumscribed, and although the overlying The majority (70%) of GISTs are spindle cell mucosa is often ulcerated, true invasion of the mucosa is lesions (Figure 5.18A–B). Of the remaining 30%, 20% rare, and is associated with a worse prognosis. are epithelioid (Figure 5.18C–D), and 10% are mixed. (A) (B) (C) (D) FIGURE 5.17 This gastric GIST can be seen arising from the muscularis propria (A). The cut surface is hemorrhagic, and there are focal areas of necrosis. This partial gastrectomy specimen shows a GIST that is roughly centered in the bowel wall, beneath the mucosa (B). There is central cystic degeneration. This large small bowel GIST shows a firm, somewhat whorled cut surface (C). GISTs may protrude into the lumen in a polypoid fashion (D), and show overlying mucosal ulceration. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 97 (A) (B) (C) (D) (E) (F) FIGURE 5.18 The majority of GISTs are spindled (A–B), and may also show nuclear palisading (A). Tumor cells are monomorphic, and lack nuclear pleomorphism (B). Approximately 20% of GISTs are epithelioid (C–D). In spindle cell tumors, nuclei are bland and elongated, with eosinophilic cytoplasm (E) and inconspicuous nucleoli. Some GISTs, especially in the stomach, have prominent perinuclear vacuoles (F). (continued) 9 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (G) (H) (I) (J) FIGURE 5.18 (continued) Stroma may be hyalinized or contain calcifications (G), and typically GISTs contain prominent vessels (H). Infrequently seen features include skeinoid fibers (arrows, I) and plasmacytoid cellular morphology (J). Cellularity is quite variable. Nuclei are usually bland, Other features that may be infrequently seen in GISTs elongated, and uniform, with pale eosinophilic cyto- include skeinoid fibers (Figure 5.18I); nuclear palisading plasm (Figure 5.18E). Nucleoli are usually inconspicu- that mimics schwannoma (Figure 5.3); and plasmacytoid ous, and perinuclear vacuoles may be seen (Figure cellular morphology (Figure 5.18J). Nuclear palisading 5.18F). As noted in the section on nuclear atypia and and cytoplasmic vacuolization are most often seen in gas- pleomorphism, the majority of GISTs are monomorphic, tric GIST, whereas skeinoid fibers are almost always found and nuclear pleomorphism is rare even in malignant in small bowel GISTs. tumors. The appearance of the stroma is also variable, The term “dedifferentiated GIST” refers to tumors and it may be myxoid or hyalinized, with variably pres- with areas of morphologically typical GIST adjacent to ent calcifications (Figure 5.18G). GISTs are typically areas of high-grade sarcoma (Figure 5.2). The sarcomatous quite vascular (Figure 5.18H) and the vessels may be component is typically KIT negative and more mitotically hyalinized. active than a typical GIST. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 9 9 (A) (B) FIGURE 5.19 Succinate dehydrogenase (SDH) deficient GISTs have distinctive histology, featuring a multinodular or plexiform growth pattern (A). Virtually all of them demonstrate a loss of SDHB staining by immunohistochemistry (B). Figures courtesy of Dr. Jason L. Hornick. A more recently described subtype of GIST is the A more recently described antibody that is very use- SDH-deficient GIST (also known as pediatric GIST ful in the diagnosis of GIST is DOG1 or ANO-1. This or type 2 GIST). These tumors often occur in female is a very sensitive and specific marker that is present in children (peak age of onset in the second decade) in the the vast majority of GISTs (Figure 5.20). The majority of stomach or omentum. The distinctive histology features KIT-negative GISTs stain with DOG1, including 79% of a multinodular or plexiform growth pattern (Figure 5.5 PDGFRA-positive GISTs (as opposed to approximately and Figure 5.19A–B), often with epithelioid or mixed 9% of PDGFRA-positive GISTs that stain with KIT). cell types. These tumors are positive for KIT, but virtu- However, as the Food and Drug Administration (FDA)- ally all demonstrate a loss of SDHB staining by immu- approved therapies for GIST require the demonstration of nohistochemistry, and thus this stain is a good screening KIT positivity, it is still necessary to confirm the diagnosis tool for this subtype of GIST. Only a minority of these with KIT. Caveats regarding the use of KIT and DOG1 tumors have an identifiable SDH mutation. Typical were discussed previously. grading criteria are not applicable to this subtype (see the section on grading and risk stratification), and they are often imatinib resistant. Additionally, this subtype is more likely to metastasize to lymph nodes, although this finding does not appear to affect prognosis. These tumors are uncommon in adults, accounting for only 8% of gastric GISTs. Immunohistochemistry The most characteristic immunophenotypic feature of GIST is KIT immunoreactivity, which may be cytoplas- mic, dot-like, or membranous (see Figure 5.8), or a mix- ture of these three patterns. Ninety-five percent of GISTs are KIT positive, and most (~99%) also mark with DOG1 and CD34. Approximately 5% of GISTs are KIT nega- tive; the majority of these are epithelioid GISTs that have platelet derived growth factor receptor A (PDGFRA) mutations, and these typically arise in the stomach or are FIGURE 5.20 Diffuse DOG1 positivity is seen in this extraintestinal (see also Chapter 13). needle biopsy from a small bowel GIST. 10 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Molecular Features the morphology of SDH-deficient GIST). Wild-type GISTs are associated with tumors seen in the pediatric popula- Approximately 80% of GISTs have KIT mutations. This tion, as well as those occurring in the setting of neurofibro- gene encodes the KIT tyrosine kinase receptor, a mem- matosis 1, Carney triad, and Carney–Stratakis syndrome. brane-associated member of the type III tyrosine kinase family that is involved in the proliferation of germ cells, Grading, Risk Stratifi cation, and Prognosis mast cells, melanocytes, and the interstitial cells of Cajal. Oncogenic KIT mutations impair regulation of receptor The most important parameters for grading and assess- activation or alter enzymatic function of tyrosine kinases. ing behavior risk in GIST are size, anatomic location, Up to 20% of tumors contain wild-type KIT, how- and mitotic rate. These criteria should only be applied to ever, and of these nearly 10% harbor mutations in primary, unifocal GIST that have not been treated with PDGFRA, or platelet-derived growth factor A. This gene either neoadjuvant or adjuvant therapy. also encodes a type III tyrosine kinase receptor that is There are multiple schemes for GIST risk stratifica- highly homologous to KIT. This subset of GISTs is more tion, including the National Institutes of Health (NIH) likely to occur in the stomach or proximal small bowel, Consensus Criteria, the Armed Forces Institute of or to be extraintestinal, and they are typically epithelioid Pathology (AFIP) modification of the NIH criteria, and with a myxoid stroma and occasional multinucleated or more recent modifications to the AFIP criteria known as rhabdoid cells. They have a lower potential for malignant the Joensuu criteria (see Table 5.6). Some authorities pre- behavior as well. Immunohistochemistry for KIT in these fer the Joensuu criteria, as they are believed to be more tumors shows faint positive or negative staining (see case accurate in identifying patients at high risk for develop- examples in the Specific Illustrative Examples section). ing recurrence or metastasis, and thus should receive adju- Certain mutations (both KIT and PDGFRA mutations) vant therapy. Mucosal invasion and rupture are associated may affect prognosis, and as a result molecular analysis with a high risk of disease progression, regardless of the of GISTs is increasingly regarded as the standard of care. site. Furthermore, if a GIST presents with metastases, this Some GISTs lack both KIT and PDGFRA mutations, tumor should be regarded as malignant, and further eval- and may be referred to as “wild-type” GIST. Some of uation is not necessary. EGISTs are evaluated according to these tumors harbor BRAF V600E mutations; these are the criteria for jejunum/ileum. KIT positive by immunohistochemistry. GISTs with BRAF Conventional GISTs rarely metastasize to lymph V600E mutations are more commonly seen in the small nodes, but when metastases occur, they are more likely to bowel, and often show more aggressive behavior. Another be in the liver or within the abdomen. Metastases to lung, subgroup of wild-type GIST shows mutations in subunits skin, and bone have been rarely described. Unlike con- A, B, C, or D of SDH, which encodes succinate dehydro- ventional GISTs, SDH-deficient GISTs do have a tendency genase. SDH mutations are mutually exclusive with other to metastasize to lymph nodes (approximately half of the GIST mutations. Approximately 42% of wild-type GIST cases), yet overall their behavior is indolent even in the demonstrate loss of succinate dehydrogenase B (SDHB) context of lymph node metastases. Because of this unusual expression by immunohistochemistry, and are known as behavior, standard criteria for grading and risk stratifi- SDH-deficient GIST (see also the previous discussion on cation are probably not applicable to this subgroup of TABLE 5.6 Risk Stratification of Primary GIST Tumor Risk of Metastasis and/ Characteristics or Death From Disease Mitotic Index Size Stomach Duodenum Jejunum/Ileum Colorectum <5/HPF <2 cm Extremely low Extremely low Extremely low Extremely low >2–< 5 cm Very low (1.9%) Low (8.3%) Low (4.3%) Low (8.5%) >5–< 10 cm Low (3.6%) Inadequate data Intermediate (24%) Inadequate data >10 cm Intermediate (10%) High (34%) High (52%) High (57%) >5/HPF < 2 cm None but few cases Inadequate data High but few cases High (57%) >2–< 5 cm Intermediate (16%) High (50%) High (73%) High (52%) >5–<10 cm High (55%) Inadequate data High (85%) Inadequate data >10 cm High (86%) High (86%) High (90%) High (71%) Source: Adapted from Downs-Kelly E, Rubin BP, Goldblum JR. Mesenchymal Tumors of the Gastrointestinal Tract. In Odze RD, Goldblum JR, Surgical Pathology of the GI Tract, Liver, Biliary Tract, and Pancreas. Philadelphia: Elsevier, 2015; Fletcher CD, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol. 2002;33:459–465; Miettinen M et al. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Sem Diag Pathol. 2006;23:70–83; Joensuu H. Risk stratification of patients diagnosed with gastrointestinal stromal tumor. Hum Pathol. 2008;39:1411–1419. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 101 tumors, and it has been recommended that conventional criteria not be used in this specific context. Therapy The first-line therapy for GIST is complete resection. Because approximately 40% of GISTs recur or metasta- size after complete resection, adjuvant therapy is common, and is based on targeting the mutant KIT or PDGFRA proteins. As previously mentioned in the section on molec- ular features, the type of mutation may predict response to therapy, and thus mutational analysis

is strongly rec- ommended for clinical management (see also Chapters 13 and 14). Imatinib mesylate, or Gleevec, is a tyrosine kinase inhibitor that targets KIT, PDGFRA, BCR-ABL, and other mutations. It is the FDA-approved first-line therapy FIGURE 5.21 Treatment-associated changes in GISTs for KIT-positive GIST patients, and it is associated with include fibrosis, decreased cellularity, and a myxoid therapeutic response rates up to 90%. Imatinib is occa- stroma. No viable tumor cells are seen in this field, sionally used to treat primary, nonmetastatic disease if the although they can usually be found on examination of tumor has a very high risk for malignant behavior. Tumors multiple sections. with exon 11 KIT mutations respond best to imatinib therapy, followed by those that are KIT/PDGFRA wild- type. GISTs with exon 9 KIT mutations may respond to a Patients with familial GISTs have germline mutations higher dose of imatinib or standard dosing with sunitinib. in either KIT or PDGFRA that are identical to sporadic Approximately 10% to 30% of GISTs show primary mutations in the same sites. The pattern of inheritance is resistance, or progression of disease within 3 to 6 months autosomal dominant, and there is approximately 100% of initiating therapy; tumors with primary resistance penetrance. Patients often have multiple tumors, and may include those with wild-type KIT, KIT exon 9 mutations, have associated hyperplasia of the interstitial cells of Cajal. and most common PDGFRA mutations. Some patients Up to 10% of patients with NF1 have GISTs as well; have what is known as delayed or secondary resistance, these tumors are often multiple and within the small meaning that the tumors show partial response or stabili- bowel. NF1-associated GISTs lack KIT and PDGFRA zation of disease, only to progress 6 months or more after mutations, and may contain skeinoid fibers histologically. the initial response or disease stabilization. This typically Carney triad includes epithelioid gastric GIST, pulmonary happens within 2 years of initiation of therapy. Resistance chondroma, and paragangliomas; GISTs in this context are may be generalized to the entire tumor or tumor burden, wild-type, and believed to be part of the spectrum of SDH- or limited to certain foci. Two of the most common muta- deficient GIST. Patients with Carney–Stratakis syndrome tions that confer resistance to imatinib are sensitive to the have epithelioid gastric GIST and paraganglioma. These drug sunitinib, which has been approved for patients who patients have germline SDH subunit gene mutations and have failed or cannot tolerate imatinib. Tumors deficient thus are also part of the spectrum of SDH-deficient GIST. in SDH are often resistant to imatinib, but may show some response to therapy with sunitinib. Differential Diagnosis Pathologists may occasionally be asked to evaluate the percentage of viable GIST in a resection specimen. The most common entities in the differential diagnosis Treatment-associated changes include a decrease in cellular- of GIST include smooth muscle tumors, schwannoma, ity, myxoid stroma, fibrosis, and necrosis, but islands of via- and desmoid tumor. The immunophenotypic comparison ble cells are usually present as well (Figure 5.21). Grading/ of these neoplasms is summarized in Table 5.4, and it is risk stratification criteria do not apply to treated GISTs. important to note that a relatively small panel of antibod- ies can address the majority of entities in the differential diagnosis. Less common mesenchymal tumors that may Genetic Syndromes Involving GIST enter into the differential diagnosis include inflamma- Although the vast majority of GISTs are sporadic, there tory fibroid polyp, IMFT, neural tumors, glomus tumor, are a number of important genetic syndromes that involve and PEComa. The differential diagnosis of GIST, includ- GISTs, including Carney triad, Carney–Stratakis syn- ing both common and uncommon entities, is discussed in drome, neurofibromatosis type 1 (NF1), and familial GIST. more detail in the sections that follow. 10 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Leiomyoma Esophageal leiomyomas are most commonly seen in the fifth decade, and some studies report a male predominance. Leiomyomas are the second most common mesenchymal tumor of the GI tract, and the most common mesenchy- Rectal tumors are also most commonly encountered in the mal tumor of the esophagus. The majority (approximately fifth to sixth decades. Leiomyomas may present incidentally, 80%) of these benign tumors present as polypoid lesions or with GI bleeding; esophageal lesions may be associated in the colon, and are often found incidentally at colonos- with a cough or dysphagia. Leiomyomas are very variable copy for some other indication. The second most com- in size, ranging from a few millimeters to 20 cm in size. mon presentation is that of an intramural mass in the Esophageal tumors most often arise from the inner layer of esophagus, most often at the gastroesophageal junction the muscularis propria, but a significant minority arise from (Figure 5.22A–B). Multiple esophageal leiomyomas are the muscularis mucosae. Colonic lesions tend to be smaller referred to as “seedling” leiomyomas or, if large, leiomyo- than esophageal leiomyomas, and arise from the muscula- matosis. Leiomyomas may also present as mural masses in ris mucosae. Grossly, these tumors are well-circumscribed, the stomach and small bowel, or in association with the unencapsulated, and often lobulated, with a firm, white to anal sphincters. tan, whorled, fibrous appearance on the cut section. (A) (B) (C) (D) FIGURE 5.22 (continued) 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 10 3 (E) (F) (G) FIGURE 5.22 This gross photograph illustrates a large tubular leiomyoma with a small fragment of overlying normal mucosa (A). Leiomyomas of the esophagus are most often at the gastroesophageal (GE) junction, as seen here (B). This small, well-circumscribed duodenal leiomyoma arises from the muscularis mucosae (C). At low power, leiomyomas are composed of fascicles of spindle cells with eosinophilic cytoplasm and elongated, cigar-shaped nuclei (D–E). Leiomyomas are diffusely positive for desmin (F), but negative for KIT (G). Histologically, leiomyomas feature fascicles of spin- and necrosis should be absent. Epithelioid leiomyomas dle cells with bright eosinophilic cytoplasm and elon- have not been described in the GI tract. gated, cigar-shaped nuclei with tapering or blunt ends Leiomyomas show immunoreactivity for SMA, des- (Figure 5.22C–E). Cytologic atypia is rare, although min, and caldesmon (Figure 5.22F–G). KIT, DOG1, and degenerative atypia has been described, similar to sym- CD34 are characteristically negative, as is S100, although plastic leiomyomas in the uterus. Hyalinization and calci- large intramural leiomyomas may rarely show KIT and/ fication may be present. Mitotic activity is low or absent, or DOG1 positivity. Mast cells within leiomyomas will, of typically less than 1 mitosis/50 high-power field (HPF), course, stain with KIT. 10 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Leiomyomatosis peritonealis disseminata is an unusual Given the S100 positivity, the differential diagnosis of condition in which multiple tumors composed of smooth schwannomas also includes melanoma (which should muscle arise beneath the peritoneum throughout the abdo- mark with other melanoma markers). men. This disease is seen almost exclusively in women of reproductive age, and many are on oral contraceptives or are pregnant, suggesting that estrogens may be a contrib- BENIGN NEURAL POLYPS uting factor. The histologic findings range from nodules or infiltrative foci of smooth muscle to a fibroblastic or Granular Cell Tumor myofibroblastic proliferation. Nuclear pleomorphism Granular cell tumors, named for their abundant granular is minimal, and mitoses are rare. Many of these lesions cytoplasm, are believed to be of neural (Schwannian) ori- regress after pregnancy or withdrawal of estrogens, and gin. The most common site in the GI tract is the esopha- malignant degeneration is rare and somewhat debatable gus, followed by the large bowel and perianal area. The in the literature. vast majority of these lesions are benign, and they rarely recur even if inadequately excised. Tumors consist of Schwannoma polygonal or spindle cells with abundant granular eosino- Gastrointestinal schwannomas are most commonly found philic cytoplasm, small uniform nuclei, and small nucleoli in the stomach, followed by the colorectum, esophagus, (Figure 5.24A–B). The spindled cell variant is most likely and small bowel. Patients are typically in the sixth or to cause confusion with GIST or other common spindle seventh decade, and gastric tumors are more common in cell lesions of the GI tract (Figure 5.24C–D). Granular women. Presenting symptoms are related to tumor loca- cell tumors are strongly and diffusely S100 positive tion, and include dysphagia with esophageal tumors; dys- (Figure 5.24E). Similar to their counterparts elsewhere, pepsia, abdominal pain, and bleeding with gastric tumors; GI granular cell tumors may have overlying pseudoepi- and bleeding or obstruction with intestinal lesions. theliomatous hyperplasia or acanthosis, which may mimic Schwannomas generally arise in the muscularis pro- squamous cell carcinoma (Figure 5.24A). pria, but they may bulge into the lumen, with overlying mucosal ulceration (Figure 5.23A). Grossly, they are cir- Ganglioneuroma/Ganglioneuromatosis cumscribed, but unencapsulated, and measure from 1 cm Ganglioneuromas (GNs) are composed of an admixture of to over 10 cm. The cut surface is homogeneous, firm, rub- Schwann cells, which are the predominant cell type, along bery, and yellow gray to tan. Cystic change and hemor- with ganglion cells and nerve fibers (Figure 5.25A–C). rhage are not usually seen. Colorectal lesions may present There are three contexts in which these lesions are found as polyps. in the GI tract. The sporadic, solitary GN is the most com- A notable feature in GI tract schwannomas (as mon; these are usually incidental findings on the left side of opposed to those that occur in the soft tissues) is a the colon, measuring less than 1.0 cm. Ganglioneuromas dense lymphoplasmacytic cuff, often with germinal cen- in the form of ganglioneuromatous polyposis are associ- ters, present at the periphery of the tumor (Figures 5.4 ated with Cowden syndrome and NF1, and diffuse gan- and 5.23B). Numerous lymphocytes may also be seen glioneuromatosis is associated with MEN-2B and NF1. admixed with tumor cells (Figure 5.23C). Schwannomas Unlike granular cell tumors, which form an expansile are moderately cellular, and are composed of spindle cells mass, ganglioneuromas entrap and surround the muco- arranged in short bundles. Focal nuclear atypia is com- sal crypts. The Schwann cell component is S100 positive mon (Figure 5.23D), but mitotic figures should be rare to (Figure 5.25D), and the nerve fibers are neurofilament absent, and atypical mitoses should not be seen. Unlike protein (NFP) positive. conventional soft tissue schwannomas, palisading is rare in the GI tract, as are foamy histiocytes. Epithelioid foci are very rare in GI tract schwannomas. Mucosal Schwann Cell Hamartoma The variant known as microcystic or reticular schwan- This is a recently described lesion that is distinct from noma may occur in the GI tract, and may mimic mucinous other neural lesions in the GI tract, including neurofi- adenocarcinoma. This variant of schwannoma also lacks broma and mucosal neuroma, which are associated with the typical lymphoid cuff, and has a striking microcystic/ syndromes and rarely encountered sporadically. These reticular growth pattern (Figure 5.23E). usually incidental lesions are typically found in the rec- All GI tract schwannomas express S100 (Figure 5.23F), tosigmoid colon of middle-aged to older adults; there is and generally lack immunoreactivity for KIT, DOG1, a female predominance. They measure less than 5 mm, SMA, desmin, and other smooth muscle markers; how- and consist of a proliferation of spindle-shaped, uniform ever, rare tumors can show focal expression of keratin, Schwann cells with plump tapering nuclei and eosinophilic SMA, or desmin. In addition, unlike their soft tissue coun- cytoplasm (Figure 5.26A–B). These lesions are purely com- terparts, GI tract schwannomas do not express calretinin. posed of Schwann cells, with no ganglion cells or axons 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 10 5 (A) (B) (C) (D) (E) (F) FIGURE 5.23 Schwannomas typically arise in the muscularis propria, and may bulge into the lumen (A, courtesy of Dr. Keisuke Goto). They are circumscribed but unencapsulated. The cut surface is yellow–tan, rubbery, and homogeneous. GI tract schwannomas have a dense peripheral lymphoplasmacytic cuff, often with lymphoid aggregates (B). Intratumoral lymphocytes are also common (C). Schwannomas are typically moderately cellular and are composed of bundles of spindle cells with focal nuclear atypia but no mitoses (D). Microcystic or reticular schwannomas may occur in the GI tract as well, and feature a striking microcystic or reticular growth pattern (E). They lack a lymphoid cuff, and may mimic mucinous adenocarcinoma. Schwannomas diffusely and strongly express S100 (F). 10 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B)

(C) (D) (E) FIGURE 5.24 Granular cell tumors consist of polygonal or spindle cells with abundant granular eosinophilic cytoplasm. Associated pseudoepitheliomatous squamous hyperplasia is common (A). Tumor cells have small uniform nuclei, small nucleoli, and abundant granular eosinophilic cytoplasm (B). The spindled variant may cause confusion with GIST, but the abundant granular cytoplasm is usually still apparent (C–D). Granular cell tumors are strongly and diffusely S100 positive (E). 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 107 (A) (B) (C) (D) FIGURE 5.25 Ganglioneuromas consist of a proliferation of Schwann cells and ganglion cells that infiltrate between the crypts in the bowel mucosa (A–C). The Schwann cell component is strongly S100 positive (D). (in contrast to ganglioneuroma). They are strongly S100 are typically sessile polypoid lesions in the rectosig- positive (Figure 5.11). Mucosal Schwann cell hamartomas moid colon, which endoscopically may be mistaken for are limited to the lamina propria, and often entrap crypts. a hyperplastic polyp or adenoma. These bland spindle cell lesions expand the lamina propria and entrap crypts (Figure 5.27A). They may have a lamellar or a whorled Perineurioma pattern, and consist of bland spindle cells with ovoid to Perineuriomas (also classified as benign fibroblastic tapering nuclei, fine fibrillary stroma, and no nuclear polyps by some authors) are usually incidental find- atypia or mitoses (Figure 5.27B). These lesions are posi- ings in middle-aged adults undergoing colonoscopy for tive for epithelial membrane antigen (EMA), although another reason. There is a female predominance. These staining may be weak and extremely focal. They are 10 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 5.26 Schwann cell hamartomas consist of a proliferation of spindle-shaped, uniform Schwann cells with plump tapering nuclei and eosinophilic cytoplasm (A–B). No ganglion cells or axons are present, in contrast to ganglioneuromas. They often infiltrate between the crypts, and are strongly S100 positive. negative for S100, and 40% to 90% express claudin-1 a variety of ways, including a mass, abdominal pain, bleed- (Figure 5.27C). Of note, perineurioma-like prolifera- ing, or signs of obstruction or fistula formation. tions frequently occur in association with both hyper- Desmoid tumors are typically large at the time of plastic polyps and sessile serrated adenomas, although resection, ranging from 4 to 25 cm. Grossly they are firm, these areas may be focal (Figure 5.27D), and some infiltrative, poorly circumscribed tumors with a coarse, tra- authors have argued that these lesions represent a mixed becular, gritty cut surface (Figure 5.29A). Histologically, stromal/epithelial polyp. Indeed, BRAF mutations are these tumors are uniform and monotonous, consisting of detected in the spindled and epithelial cell components long, sweeping, broad fascicles of slender spindle-shaped of polyps with both elements. cells with elongated nuclei and eosinophilic cytoplasm A wide variety of neural tumors are associated with (Figure 5.29B–D). Nuclei have fine chromatin and incon- spicuous nucleoli. Mitotic activity is variable but usually neurofibromatosis (NF1), including ganglioneuromatous low, and atypical mitoses should be absent. Vessels are polyposis, diffuse ganglioneuromatosis, neurofibromas typically prominent and may be ectatic or compressed (Figure 5.28A–B), diffuse neuromatosis, and gangliocytic between fascicles of tumor cells. Some tumors contain paraganglioma. As previously mentioned in the section myxoid areas, dense (keloidal) hyalinization, fasciitis-like on genetic syndromes involving GISTs, NF1 patients also areas, or areas with a loose, storiform growth pattern. have an increased incidence of GIST. Unlike some of the other entities in the differential diagnosis of GIST, these tumors arise in the mesentery and Desmoid Tumor (Intra-Abdominal invade the bowel, rather than the reverse. However, they Desmoid Fibromatosis) can be deeply infiltrative, extending all the way through the wall to the mucosa. Tumor cells express nuclear Intra-abdominal desmoid tumors primarily affect the mes- β-catenin (Figure 5.15) in most cases, and tumor cells are entery or retroperitoneum, but when these lesions invade or negative for DOG1, S100, desmin, and caldesmon. The encroach upon the bowel wall, they may mimic a primary vast majority of desmoid tumors are KIT negative, but GI mesenchymal neoplasm (particularly GIST). Tumors positive staining has been reported very rarely. may occur in the abdomen or in the pelvis. Of note, 25% Desmoid tumors lack the potential for metastasis, but occur in the context of familial adenomatous polyposis/ have a high likelihood of local recurrence, and the clinical Gardner syndrome. These tumors may present at any age, course is often unpredictable. Surgery is the mainstay of and there is no gender predilection. Patients can present in therapy. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 10 9 (A) (B) (C) (D) FIGURE 5.27 Perineuriomas are composed of a proliferation of bland spindle cells that expand the lamina propria and entrap crypts (A). The cells have ovoid to tapering nuclei, fine fibrillary stroma, and no nuclear atypia or mitoses (B). Many of these lesions stain with claudin-1 (C). Perineurioma-like proliferations frequently occur in association with both hyperplastic polyps and sessile serrated adenomas (D). Sclerosing mesenteritis may also enter into the differ- β-catenin staining is not seen in sclerosing mesenteritis. ential diagnosis of either GIST or desmoid tumor. This Of note, sclerosing mesenteritis has been rarely reported idiopathic disease, also known as mesenteric panniculi- to express KIT. tis, liposclerotic mesenteritis, mesenteric Weber–Christian disease, xanthogranulomatous mesenteritis, mesenteric lipogranuloma, systemic nodular panniculitis, inflamma- LESS COMMON/RARE MESENCHYMAL tory pseudotumor, or mesenteric lipodystrophy is a fibro- TUMORS OF THE GI TRACT inflammatory disorder that primarily affects the small bowel mesentery. It most often presents as a large, unifocal Glomus Tumor mass, although approximately 20% of patients have mul- Glomus tumors are most commonly found in the skin and tiple lesions. These lesions consist of fibrous bands that infiltrate and encase fat lobules, with associated inflam- subcutis of the distal extremities, but they occasionally mation (predominantly mononuclear cells) and fat necro- arise in the GI tract, most commonly in the antrum of the sis. The presence of inflammation and fat necrosis helps stomach. Patients are from a wide age range, and there is a differentiate these from desmoid tumors, and nuclear female predominance in some series. Tumors may present 110 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 5.28 This patient with NF1 had multiple gastrointestinal plexiform neurofibromas, such as the colonic one shown here (A–B). (A) (B) (C) (D) FIGURE 5.29 This desmoid tumor forms a large, homogeneous mass with a trabecular cut surface and adherent loops of bowel (A, courtesy of Dr. Petur Nielsen). These tumors contain a monotonous population of spindle cells arranged in long, sweeping, broad fascicles with prominent vessels (B). Cells are slender with elongated nuclei, fine chromatin, inconspicuous nucleoli, and eosinophilic cytoplasm (C–D). 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 111 with upper GI bleeding, abdominal pain, or as inciden- is typically diffuse reactivity for SMA (Figure 5.30D), but tal findings on evaluation for something unrelated. The KIT and DOG1 are negative, unlike GIST. Calponin and majority of tumors are benign, but rare malignant glomus caldesmon are typically positive, but desmin is often nega- tumors have been reported. tive, in contrast to smooth muscle tumors. Some tumors Glomus tumors are typically 1 to 3 cm, arise in the are focally positive with CD34 and synaptophysin, but muscularis propria, and feature cellular nodules or nests chromogranin and keratin should be negative. Pericellular separated by bands of smooth muscle extending from the membranous positivity is seen with laminin and collagen muscularis propria (Figure 5.30A–C). Prominent slit-like type IV. and dilated vessels are common, and the tumor cells show Other vascular tumors occurring in the gut that may a subendothelial growth pattern within walls of blood have spindle cell morphology include angiosarcoma and vessels. The cells are uniform and rounded, with clear to Kaposi’s sarcoma. If the latter is suspected, HHV-8 and vas- eosinophilic cytoplasm and sharply defined cell borders. cular markers are very helpful, but it is important to be aware The nucleus appears “punched out” and round, and is that Kaposi’s sarcoma will stain with KIT. Angiosarcoma well demarcated from the surrounding cytoplasm. There occurs very rarely in the GI tract (see also Chapter 9). (A) (B) (C) (D) FIGURE 5.30 This gastric glomus tumor is arising from the muscularis propria, and the nodules of tumor cells are surrounded by bands of smooth muscle. Prominent dilated and/or slit-like vessels are common (B). Tumor cells are uniform and round, with clear to eosinophilic cytoplasm (C, courtesy of Dr. Andrew Folpe). Reactivity for SMA is characteristic (D, courtesy of Dr. Andrew Folpe). 112 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Infl ammatory Fibroid Polyp “epithelioid inflammatory myofibroblastic sarcoma”) is highly aggressive and is characterized by epithelioid cells Inflammatory fibroid polyps are benign tumors that are with prominent nucleoli, a unique nuclear membrane or most commonly found in the antrum of the stomach (see perinuclear staining pattern of ALK, and a distinctive also Chapter 8) or the ileum (see also Chapter 9), and often RANBP2–ALK gene fusion. present as a polyp. They present in a wide age range but IMFTs are positive for SMA, and variably positive are typically seen in adults from 60 to 80 years old, and for desmin. Approximately 30% are immunopositive may be associated with intussusception. These lesions arise with keratin as well. Unlike GIST, they are negative for from the submucosa, and the overlying mucosa is often KIT and DOG1, as well as S100. Approximately half of ulcerated (Figure 5.31A). Tumors range in size from less tumors have an ALK gene rearrangement; the younger the than 0.5 cm to over 4.0 cm, with an average size of 1 to patient, the more likely the tumor is to have this altera- 2 cm. Histologically, they have ill-defined margins (Figure tion. There is imperfect correlation between the gene 5.31B), and consist of a bland proliferation of spindle rearrangement and ALK immunohistochemical positivity cells and stellate cells with prominent admixed inflam- (Figure 5.32D), however. ALK staining is not specific to matory cells, especially eosinophils (Figure 5.31C). Blood IMFT, and positivity has been reported in rhabdomyo- vessels are typically prominent, and may show surround- sarcomas and malignant peripheral nerve sheath tumors, ing “onion-skin” fibrosis (Figure 5.31D). If the surface is although GISTs are ALK negative. ulcerated, bizarre pleomorphic stromal cells can be seen, which should not be interpreted as a feature of malig- nancy. Occasionally, prominent multinucleated giant cells PEComa are present (Figure 5.31E). IFPs are positive for CD34 PEComas, or perivascular epithelial cell tumors, are a and variably positive for SMA; they are negative for KIT, family of related mesenchymal tumors that includes desmin, and S100. The neoplastic nature of these lesions angiomyolipoma, lymphangiomyomatosis, and clear cell has been debated, but some IFPs have been found to have “sugar” tumor of lung. All share a distinctive perivascular activating mutations in PDGFRA, so they in fact may be epithelioid cell phenotype, with evidence of both smooth neoplastic. Giant fibrovascular polyps of the esophagus are muscle and melanocytic differentiation. There is no known histologically somewhat similar lesions, although they lack normal tissue counterpart. These tumors typically present the eosinophilic component of IFP; these will be discussed in women of middle age, and can be found in the abdo- in Chapter 7. men/pelvis, retroperitoneum, tubal gut, uterus, liver, and kidney. Rarely, they are associated with tuberous sclero- Infl ammatory Myofi broblastic Tumor sis. Histologically, tumors are spindled and/or epithelioid, with clear to granular eosinophilic “stringy” cytoplasm IMFTs are most often seen in children and young adults. (Figure 5.33A–B). Admixed prominent blood vessels may Historically, these have also been known as inflammatory be seen, as well as adipocytes. In some cases, alveolar pseudotumors of the GI tract, or plasma cell granulomas. nests of tumor cells are surrounded by delicate vascula- They are considered lesions of intermediate risk, as they ture. Nuclear hyperchromasia and pleomorphism may have a tendency to recur but rarely metastasize. Outside be seen, and occasional cases have shown signet ring-like of the lung, the most common sites include abdomen (mes- cells, strap-like cells, and multivacuolated cells resembling entery, omentum, and tubal gut), pelvis, and retroperito- lipoblasts. Mitoses may be present but are typically not neum. Patients typically present with abdominal pain and/ numerous, and necrosis may also be seen. Rare malignant or mass, and some have prominent systemic complaints PEComas have been reported. including fever, weight loss, and malaise. PEComas, regardless of the site, are usually positive IMFTs are variably sized

at presentation, with a for SMA, and often for HMB-45 (Figure 5.33C), melan- mean of 8 to 10 cm, and are typically solitary and mul- A, and MiTF. Desmin immunoreactivity is variably tinodular. Several patterns have been described, includ- present. Some tumors, especially epithelioid or clear cell ing spindle cell lesions with myxoid or hyalinized stroma, variants, may lack SMA staining, however. There is focal more storiform or fascicular growth patterns, and hypo- S100 positivity in less than 10%. The presence of a spindle cellular sclerotic lesions. The spindle cells typically have cell component, and the fact that the majority of PEComas ovoid to tapering nuclei and pale eosinophilic cytoplasm mark with KIT, may lead to confusion with GIST. (Figures 5.32A–B). Approximately half contain ganglion- like cells (Figure 5.32C), and a prominent lymphoplasma- cytic inflammatory infiltrate is often present. Calcifications Plexiform Fibromyxoma (Plexiform Angiomyxoid and metaplastic bone may also be seen. Some tumors have Myofi broblastic Tumor of Stomach) striking cytologic atypia (Figure 5.32C). In general, there These tumors are nearly exclusive to the gastric antrum, is poor correlation between the histologic features and where they present as a mural mass in young to middle- behavior; however, a recently described variant (termed aged adults that is often mistaken for GIST. These bland, 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 113 (A) (B) (C) (D) (E) FIGURE 5.31 This inflammatory fibroid polyp of the ileum arises from the submucosa and protrudes into the lumen of the bowel (A). The interface between tumor and normal tissue is ill-defined, and may contain lymphoid aggregates. These lesions consist of a bland proliferation of spindle cells with prominent admixed inflammatory cells, especially eosinophils (B–C). Blood vessels are typically prominent, and may show surrounding “onion-skin” fibrosis (D). Occasionally, multinucleate giant cells are present (E, arrow). multilobular spindle cell tumors are sharply circumscribed, tumors are positive for SMA and variably with desmin; they have a plexiform growth pattern, and contain abun- are negative for KIT, DOG1, and S100. Plexiform fibro- dant myxoid stroma with prominent small blood vessels myxomas are rare, and thus knowledge of natural history (Figure 5.34A–B). Vascular invasion is common. These is limited, but they appear to behave in a benign fashion. 114 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 5.32 This solitary IMFT in the colon consists of interlacing bundles of spindled cells with an admixed prominent inflammatory infiltrate containing numerous plasma cells (A–B). The spindle cells typically have ovoid to tapering nuclei and pale eosinophilic cytoplasm. Approximately half contain ganglion-like cells, and there may be significant nuclear atypia (C). ALK positivity is often seen in IMFT, but is not specific for these tumors (D, courtesy of Dr. Andrew Folpe). Gangliocytic Paraganglioma Leiomyosarcoma Gangliocytic paragangliomas are triphasic tumors com- This is a rare smooth muscle tumor, accounting for only posed of a mixture of epithelioid and spindle cells along about 1% of GI mesenchymal tumors. This tumor can with ganglion cells (see also Chapters 3 and 9). They have occur anywhere in the GI tract, and symptoms vary a predilection for the second part of the duodenum. They with location. They can present as polypoid lesions, or typically present as polypoid submucosal masses, and they involve the entire thickness of the wall of the bowel. The may be associated with NF1. Histologically, tumors con- overlying mucosa may be ulcerated, as mucosal invasion sist of anastomosing cords and nests of spindled cells with by tumor is typical. Tumors are grossly fleshy masses admixed nests and clusters of epithelioid (neuroendocrine) with necrosis and hemorrhage. They are histologically cells, ganglion cells, and occasionally neuroendocrine cells identical to leiomyosarcomas elsewhere, featuring highly (Figure 5.35A–B). Tumor cells stain with chromogranin A, cellular fascicles of spindle cells containing elongated and spindled cells will stain with S100. These tumors typi- nuclei and brightly eosinophilic cytoplasm (Figure 5.36). cally behave in a benign fashion. Nuclear pleomorphism is usually prominent, and 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 115 (A) (B) (C) FIGURE 5.33 This colonic PEComa consists of a proliferation of epithelioid cells with clear to granular eosinophilic cytoplasm (A–B, courtesy of Dr. Andrew Folpe). Admixed prominent blood vessels may be seen, as well as adipocytes. Nuclear hyperchromasia and pleomorphism are present as well. HMB-45 is characteristically positive (C, courtesy of Dr. Andrew Folpe). mitotic activity is brisk, typically around 50/50 HPF, sarcoma-like tumor of the GI tract (malignant GI neuro- with atypical mitoses. Unlike GISTs, leiomyosarcomas ectodermal tumor), synovial sarcoma, alveolar soft parts are negative for KIT and DOG1, and routinely show sarcoma, rhabdomyosarcoma, and other small round blue strong SMA expression. Desmin expression varies and cell tumors such as desmoplastic small round blue cell may be lost in some higher grade tumors. Because these tumor. tumors are so rare, and many previous series mistakenly included high-grade GISTs, clinical behavior has not been well-defined. SPECIFIC ILLUSTRATIVE EXAMPLES True primary liposarcomas (LPSs) of the bowel are exceedingly rare, but dedifferentiated LPS, the most com- Case 1 mon sarcoma of the retroperitoneum, often involves the A 46-year-old woman presented with vague epigastric tubal gut by invading into the wall from an external loca- pain and nausea. Upper endoscopy was essentially nor- tion (Figure 5.37). mal, but CT scan revealed a 4 cm tumor in the body of Other unusual mesenchymal tumors that rarely occur the stomach. The patient underwent partial gastrec- in the GI tract include solitary fibrous tumor, clear cell tomy, and a firm, fleshy, white–tan tumor was resected 116 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 5.34 This gastric antral plexiform fibromyxoma is a bland, multilobular spindle cell lesion with a sharply circumscribed border and myxoid stroma (A). Small blood vessels are prominent (B). that appeared to be arising from the muscularis propria. epithelioid GIST from the stomach, with myxoid stroma, The tumor had epithelioid morphology (Figure 5.38A), suggests that this tumor may well have a PDGFRA muta- with myxoid stroma and a very low mitotic count. Upon tion, and thus mutational analysis would be useful as this immunohistochemical staining, however, the tumor was subset of GISTs has a lower potential for malignant behavior. essentially KIT negative (Figure 5.38B), but DOG1 was strongly and diffusely positive (Figure 5.38C). Case 2 This tumor emphasizes that the majority of KIT- negative GISTs mark with DOG1, including almost 80% A 30-year-old woman underwent a CT scan as part of a of PDGFRA-positive GISTs. The fact that this tumor is an workup for chronic pelvic pain. A 2 cm mass was found in (A) (B) FIGURE 5.35 Gangliocytic paragangliomas consist of cords and nests of spindled and epithelioid cells (A) with admixed ganglion cells (B, arrows). 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 117 (A) FIGURE 5.36 Gastrointestinal leiomyosarcomas are similar to their counterparts elsewhere, with highly cellular fascicles of spindle cells containing elongated nuclei with prominent pleomorphism, brightly eosinophilic cytoplasm, and brisk mitotic activity. the colon, which was subsequently resected. The tumor was composed of plump eosinophilic cells with focal nuclear pleomorphism (Figure 5.39A–B), admixed with cells with clear cytoplasm. An initial panel of immunostains showed that the tumor was KIT positive, and thus it was initially thought to be a pleomorphic GIST. However, additional (B) (C) FIGURE 5.38 This gastric GIST has prominent epithelioid morphology with abundant myxoid stroma and a very low mitotic count (A, courtesy of Dr. Jason Hornick). It is also essentially KIT negative (B, courtesy of Dr. Jason Hornick) but strongly and diffusely DOG1 positive (C, courtesy of Dr. Jason Hornick). The epithelioid morphology, gastric FIGURE 5.37 This retroperitoneal liposarcoma extends location, and myxoid stroma suggest that this tumor may into the wall of the colon. Note the overlying melanosis coli. have a PDGFRA mutation. 118 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 5.39 This colonic PEComa is composed of plump eosinophilic cells with nuclear pleomorphism and prominent nucleoli (A, courtesy of Dr. Andrew Folpe). Other cells have clear to granular cytoplasm (B, courtesy of Dr. Andrew Folpe). It would be unusual for a GIST to have this degree of nuclear pleomorphism, stains revealed that the tumor was negative for DOG1, and Gill AJ, Chou A, Vilain R, et al. Immunohistochemistry for SDHB positive for HMB-45 and SMA, characteristic of a PEComa. divides gastrointestinal stromal tumors into 2 distinct types. Am J Surg Pathol. 2010;34:636–644. This case illustrates the confusion that may arise Joensuu H, Roberts PJ, Sarlomo-Rikala M, et al. Effect of the tyrosine between PEComa (especially those with a prominent spin- kinase inhibitor STI571 in a patient with a metastatic gastrointes- dle cell component) and GIST, due to the fact that both tinal stromal tumor. N Engl J Med. 2001;344:1052–1056. are KIT positive. As previously discussed in the section on Lasota J, Dansonka-Mieszkowska A, Sobin LH, Miettinen M. A great majority of GISTs with PDGFRA mutations represent PEComa, additional immunostains are helpful in resolv- gastric tumors of low or no malignant potential. Lab Invest. ing the differential diagnosis. In addition, it is unusual for 2004;84:874–883. a GIST to have this degree of nuclear pleomorphism and Miettinen M, Killian JK, Wang ZF, et al. Immunohistochemical loss prominence of nucleoli. of succinate dehydrogenase subunit A (SDHA) in gastrointestinal stromal tumors (GISTs) signals SDHA germline mutation. Am J Surg Pathol. 2013;37:234–240. Miettinen M, Lasota J. Histopathology of gastrointestinal stromal SELECTED REFERENCES tumor. J Surg Oncol. 2011;104:865–873. Miettinen M, Sarloma-Rikala M, Sobin LH, Lasota J. Esophageal Gastrointestinal Stromal Tumors stromal tumors: a clinicopathologic, immunohistochemical, and molecular genetic study of 17 cases and comparison with Abraham SC, Krasinskas AM, Hofstetter WL, et al. “Seedling” mesen- esophageal leiomyomas and leiomyosarcomas. Am J Surg Pathol. chymal tumors (gastrointestinal stromal tumors and leiomyomas) 2000;24:211–222. are common incidental tumors of the esophagogastric junction. Miettinen M, Sobin LH, Lasota J, Gastrointestinal stromal tumors Am J Surg Pathol. 2007;31:1629–1635. of the stomach. A clinicopathologic, immunohistochemical, and Antonescu CR, Romeo S, Zhang L, et al. Dedifferentiation in gastroin- molecular genetic study of 1765 cases with long term follow up. testinal stromal tumor to an anaplastaic KIT-negative phenotype: Am J Surg Pathol. 2005;29:52–68. a diagnostic pitfall. Am J Surg Pathol. 2013;37:385–392. Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors pre- Carvalho N, Albergaria D, Lebre R, et al. Anal canal gastrointes- senting as omental masses: a clinicopathologic analysis of 95 cases. tinal stromal tumors: case report and literature review. World J Am J Surg Pathol. 2009;33:1267–1275. Gastroenterol. 2014;20:319–322. Miettinen M, Wang ZF, Sarlomo-Rikala M, et al. Succinate Corless CL. Gastrointestinal stromal tumors: what do we know now? dehydrogenase-deficient GISTs: a clinicopathologic, immu- Mod Pathol. 2014;27:S1–S16. nohistochemical, and molecular genetic study of 66 gas- Dematteo RP, Gold JS, Saran L, et al. Tumor mitotic rate, size, tric GISTs with predilection to young age. Am J Surg Pathol. and location independently predict recurrence after resec- 2011;35(11):1712–1721. tion of primary gastrointestinal stromal tumor (GIST). Cancer. Miettinien M, Sarlomo-Rikala M, Lasota J. Gastrointestinal stromal 2008;112(3):608–615. tumors: recent advances in understanding of their biology. Hum Doyle LA, Nelson D, Heinrich MC, et al. Loss of succinate dehydro- Pathol. 1999;30:1213–1220. genase subunit B (SDHB) expression is limited to a distinctive Patil DT, Rubin BP. Gastrointestinal stromal tumor: advances in diagno- subset of gastric wild-type gastrointestinal stromal tumors: a com- sis and management. Arch Pathol Lab Med. 2011;135:1298–1310. prehensive genotype-phenotype correlation study. Histopathol. Wagner AJ, Remillard SP, Zhang YX, et al. Loss of expression of SDHA 2012;61:801–809. predicts SDHA mutations in gastrointestinal stromal tumors. Mod Dwight T, Benn DE, Clarkson A, et al. Loss of SDHA expres- Pathol. 2013;26:289–294. sion identifies SDHA mutations in succinate dehydrogenase- Yantiss RK, Rosenberg AE, Sarran L, et al. Multiple gastrointestinal deficient gastrointestinal stromal tumors. Am J Surg Pathol. stromal tumors in type I neurofibromatosis: a pathologic and 2013;37:226–233. molecular study. Mod Pathol. 2005;18:475–484. 5 Approach to Mesenchymal Neoplasms of the Gastrointestinal Tract 119 Immunohistochemistry Miettinen M, Furlong M, Sarloma-Rikala M, et al. Gastrointestinal stromal tumors, intramural leiomyomas, and leimyosarcomas in Espinosa I, Lee CH, Kim MK, et al. A novel monoclonal antibody the rectum and anus. A clinicopathologic, immunohistochemi- against DOG1 is a sensitive and specific marker for gastrointesti- cal, and molecular genetic study of 144 cases. Am J Surg Pathol. nal stromal tumors. Am J Surg Pathol. 2008;32:210–218. 2001;25:1121–1133. Guler ML, Daniels JA, Abraham SC, Montgonery EA. Melanoma anti- Miettinen M, Sarloma-Rikala

M, Sobin LH. Mesenchymal tumors of gens in epithelioid gastrointestinal stromal tumors: a potential muscularis mucosa of colon and rectum are benign leiomyomas diagnostic pitfall. Arch Pathol Lab Med. 2008;132:1302–1306. that should be separated from gastrointestinal stromal tumors–a Hemminger J, Iwenofu OH. Discovered on gastrointestinal stromal clinicopathological and immunohistochemical study of eighty- tumours 1(DOG1) expression in non-gastrointestinal stromal eight cases. Mod Pathol. 2001;14:950–956. tumour (GIST) neoplasms. Histopathol. 2012; 61:170–177. Mutrie CJ, Donahue DM, Wain JC, et al. Esophageal leiomyoma: a Hornick JL. Novel uses of immunohistochemistry in the diagnosis and 40-year experience. Ann Thorac Surg. 2005;79:1122–1125. classification of soft tissue tumors. Mod Pathol. 2014;27:S47–S63. Yamamoto H, Handa M, Tobo T, et al. Clinicopathologic features Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and of primary leiomyosarcoma of the gastrointestinal tract follow- prognosis at different sites. Semin Diag Pathol. 2006;23:70–83. ing recognition of gastrointestinal stromal tumors. Histopathol. Miettinen M, Lasota J. KIT (CD117): a review on expression in normal and 2013;63:194–207. neoplastic tissues, and mutations and their clinicopathologic correla- tion. Appl Immunohistochem Mol Morphol. 2005;13(3):205–220. Miettinen M, Want ZF, Lasota J. DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 Schwannoma cases. Am J Surg Pathol. 2009;33:1401–1408. Hou YY, Tan YS, Xu JF, et al. Schwannoma of the gastrointestinal West RB, Corless CL, Chen X, et al. The novel marker, DOG1, is tract: a clinicopathological, immunohistochemical, and ultrastruc- expressed ubiquitously in gastrointestinal stromal tumors irre- tural study of 33 cases. Histopathol. 2006;48:536–545. spective of KIT or PDGFRA mutation status. Am J Pathol. Leigl B, Bennett MW, Fletcher CDM. Microcystic/reticular schwan- 2004;165:107–113. noma: a distinct variant with predilection for visceral locations. Am J Surg Pathol. 2008;32:1080–1087. Molecular Evaluation Voltaggio L, Murray R, Lasota J, Miettinen M. Gastric schwannoma: a clinicopathologic study of 51 cases and critical review of the litera- Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal ture. Hum Pathol. 2012;43:650–659. tumors: origin and molecular oncology. Cancer. 2011;11:865–878. Corless CL, Schroeder A, Griffith D, et al. PDGFRA mutations in gas- trointestinal stromal tumors: frequency, spectrum, and in vitro Benign Neural Polyps sensitivity to imatinib. J Clin Oncol. 2005;23:5357–5364. Agaimy A, Vassos N, Croner RS. Gastrointestinal manifestations of Heinrich MC, Corless CL, Blanke CD, et al. Molecular correlates neurofibromatosis type 1 (Recklinghausens’s disease): clinicopath- of imatinib resistance in gastrointestinal stromal tumors. J Clin ologic spectrum with pathogenetic considerations. Int J Clin Exp Oncol. 2006;24:4764–4774. Pathol. 2012;5:852–862. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activat- Eslami-Varzaneh F, Washington K, Robert ME, et al. Benign fibroblas- ing mutations in gastrointestinal stromal tumors. Science. tic polyps of the colon: a histologic, immunohistochemical, and 2003;299:708–710. ultrastructural study. Am J Surg Pathol. 2004;28:374–378. Heinrich MC, Owzar K, Corless CL, et al. Correlation of kinase gen- Fuller CE, Williams GT. Gastrointestinal manifestations of type 1 neu- otype and clinical outcome in the North American Intergroup rofibromatosis (von Recklinghausen’s disease). Histopathol. Phase II trial of imatinib mesylate for treatment of advanced gas- 1991;19:1–11. trointestinal stromal tumor: CALGB 150105 study by Cancer and Gibson JA, Hornick JL. Mucosal Schwann cell “hamartoma:” clini- Leukemia Group B and Southwest Oncology Group. J Clin Oncol. copathologic study of 26 neural colorectal polyps distinct from 2008;26:5360–5367. neurofibromas and mucosal neuromas. Am J Surg Pathol. Hostein I, Faur N, Primois C, et al. BRAF mutation status in gastro- 2009;33:781–787. intestinal stromal tumors. Am J Clin Pathol. 2010;133:141–148. Groisman GM, Hershkovitz D, Vieth M, Sabo E. Colonic perineurio- Lasota J, Dansonka-Mieszkowska A, Sobin LH, Miettinen M. mas with and without crypt serrations: a comparative study. Am J A great majority of GISTs with PDGFRA mutations represent Surg Pathol. 2013;37:745–751. gastric tumors of low or no malignant potential. Lab Invest. Groisman GM, Polak-Charcon S. Fibroblastic polyp of the colon and 2004;84:874–883. colonic perineurioma: 2 names for a single entity? Am J Surg Lasota J, Stachura J, Miettinen M. GISTs with PDGFRA exon 14 muta- Pathol. 2008;32, 1088–1094. tions represent subset of clinically favorable gastric tumors with Hornick JL, Fletcher CDM. Intesitnal perineuriomas: clinicopathologic epithelioid morphology. Lab Invest. 2006;86:94–100. definition of a new anatomic subset in a series of 10 cases. Am J Oudijk L, Gaal J, Korpershoek E, et al. SDHA mutations in adult and Surg Pathol. 2005;29:859–865. pediatric wild-type gastrointestinal stromal tumors. Mod Pathol. Johnston J, Helwig EB. Granular cell tumors of the gastrointesti- 2013;26:456–463. nal tract and perianal region: a study of 74 cases. Dig Dis Sci. Plesec TP. Gastrointestinal mesenchymal neoplasms other than gas- 1981;26:807–816. trointestinal stromal tumors: focusing on their molecular aspects. Lee NC, Norton JA. Multiple endocrine neoplasia type 2B-genetic basis Pathol Res Intl. 2011;2011:1–10. and clinical expression. Surg Oncol. 2000;9:111–118. Pai RK, Mojtahed A, Rouse RV, et al. Histologic and molecular anal- Smooth Muscle Tumors yses of colonic perineurial-like proliferations in serrated polyps: perineurial-like stromal proliferations are seen in sessile serrated Bisceglia M, Galliani CA, Pizzolitto S, et al. Selected case from the adenomas. Am J Surg Pathol. 2011 35(9):1373–1380. Arkadi M. Rywlin International Pathology Slide Series: leiomyo- Shekitka KM, Sobin LH. Ganglioneuromas of the gastrointestinal matosis peritonealis disseminata: report of 3 cases with extensive tract. Relation to von Recklinghausen’s disease and other multiple review of the literature. Adv Anat Pathol. 2014;21:201–215. tumor syndromes. Am J Surg Pathol. 1994;18:250–257. 12 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Singhi AD, Montgomery EA. Colorectal granular cell tumor: a clinico- PEComa pathologic study of 26 cases. Am J Surg Pathol. 2010;34:1186–1192. Bleeker JS, Quevedo JF, Folpe AL. “Malignant” perivascular epithe- lioid cell neoplasm: risk stratification and treatment strategies. Desmoid Tumors Sarcoma. 2012; 2012:541626. Doyle LA, Hornick JL, Fletcher CDM. PEComa of the gastrointestinal Montgomery E, Torbenson MS, Kaushal MK, et al. Beta catenin immu- tract: clinicopathologic study of 35 cases with evaluation of prog- nohistochemistry separates mesenteric fibromatosis from gastro- nostic parameters. Am J Surg Pathol. 2013;37:1769–1782. intestinal stromal tumor and sclerosing mesenteritis. Am J Surg Maluf H, Dieckgraefe B. Angiomyolipoma of the large intestine: report Pathol. 2002;26;1296–1301. of a case. Mod Pathol. 1999;12:1132–1136. Yantiss RK, Spiro IJ, Compton CC, Rosenberg AE. Gastrointestinal Ryan P, Nguyen VH, Gholoum S, et al. Polypoid PEComa in the rectum stromal tumor versus intra-abdominal fibromatosis of the bowel of a 15-year-old girl. Case report and review of PEComa in the wall: a clinically important differential diagnosis. Am J Surg gastrointestinal tract. Am J Surg Pathol. 2009;33:475–482. Pathol. 2000; 24:947–957. Yamamoto H, Oda Y, Tao T, et al. Malignant perivascular epithelioid cell tumor of the colon: report of a case with molecular analysis. Path Intl. 2006;56:46–50. Glomus Tumor Kang G, Park HJ, Kim JY, et al. Glomus tumor of the stomach: a clini- Plexiform Fibromyxoma copathologic analysis of 10 cases and review of the literature. Gut Liver. 2012;6:52–57. Miettinen M, Makhlouf HR, Sobin LH, Lasota J. Plexiform fibromyx- Miettinen M, Paal E, Lasota J, Sobin LH. Gastrointestinal glomus tumors: oma: a distinctive benign gastric antral neoplasm not to be con- a clinicopathologic, immunohistochemical, and molecular genetic fused with a myxoid GIST. Am J Surg Pathol. 2009;33:1624–1632. study of 32 cases. Am J Surg Pathol. 2002;26:301–311. Takahashi Y, Shimizu S, Ishida T, et al. Plexiform angiomyx- oid myofibroblastic tumor of the stomach. Am J Surg Pathol. 2007;31:724–728. Infl ammatory Fibroid Polyp Gangliocytic Paraganglioma Lasota J, Wang ZF, Sobin LH, Miettinen M. Gain-of-function PDGFRA mutations, earlier reported in gastrointestinal stromal tumors, are Hamid QA, Bishop AE, Rode J, et al. Duodenal gangliocytic paragan- common in small intestinal inflammatory fibroid polyps. A study glioma: a study of 10 cases with immunocytochemical neuroendo- of 60 cases. Mod Pathol. 2009; 22(8):1049–1056. crine markers. Hum Pathol. 1986;17:1151–1157. Liu TC, Lin MT, Montgomery EA, Singhi AD. Inflammatory fibroid Okubo Y, Yokose T, tuchiya M, et al. Duodenal gangliocytic paragan- polyps of the gastrointestinal tract: spectrum of clinical, mor- glioma showing lymph node metastasis: a rare case report. Diagn phologic, and immunohistochemistry features. Am J Surg Pathol. Pathol. 2012;5:27. 2013;37(4):586–592. Scheithauer BW, Nora FE, Lechago J, et al. Duodenal gangliocytic para- Ozolek JA, Sasatomi E, Swalsky PA, et al. Inflammatory fibroid pol- ganglioma. Clinicopathologic and immunocytochemical study of yps of the gastrointestinal tract: clinical, pathologic, and molec- 11 cases. Am J Clin Pathol. 1986;58:1720–1735. ular characteristics. Appl Immunohistochemist Mol Morph. 2004;12:59–66. Others Akram S, Pardi DS, Schaffner JA, Smyrk TC. Sclerosing mesenteri- Infl ammatory Myofi broblastic Tumor tis: clinical features, treatment, and outcome in 92 patients. Clin Gastroenterol Hepatol. 2007;5:589–596. Coffin CM, Watterson J, Priest JR, et al. Extrapulmonary inflamma- Company Campins MM, Morales R, Dolz C, et al. Primary monophasic tory myofibroblastic tumor (inflammatory pseudotumor). A clini- synovial sarcoma of the duodenum confirmed by cytogenetic anal- copathologic and immunohistochemical study of 84 cases. Am J ysis with demonstration of t(X;18); a case report. J Gastrointestin Surg Pathol. 1995;19:859–872. Liver Dis. 2009;18:89–93. Cook JR, Dehner LP, Collins MH, et al. Anaplastic lymphoma kinase Emory TS, Monihan JM, Carr NJ, et al. Sclerosing mesenteritis, mes- (ALK) expression in the inflammatory myofibroblastic tumor: enteric panniculitis, and mesenteric lipodystrophy: a single entity? a comparative immunohistochemical study. Am J Surg Pathol. Am J Surg Pathol. 1997;21:392–398. 2001;25:1364–1371. Liu YQ, Yue JQ. Intramural solitary fibrous tumor of the ileum: Makhlouf HR, Sobin LH. Inflammatory myofibroblastic tumors a case report and review of the literature. J Cancer Res Ther. (inflammatory pseudotumors) of the gastrointestinal tract: how 2013;9:724–726. closely are they related to inflammatory fibroid polyps? Hum Lyle PL, Amato CM, Fitzpatrick JE, Robinson WA. Gastrointestinal Pathol. 2002;33:307–315. melanoma or clear cell sarcoma? Molecular evaluation of 7 cases Marino-Enriquez A, Wang WL, Roy A, et al. An aggressive intra- previously diagnosed as malignant meloma. Am J Surg Pathol. abdominal variant of inflammatory myofibroblastic tumor 2008;32:858–866. with nuclear membrane or perinuclear ALK. Am J Surg Pathol. Stockman DL, Miettinen M, Suster S, et al. Malignant gastrointestinal 2011;35:135–144. neuroectodermal tumor: clinicopathologic, immunohistochemical, Sanders BM, West KW, Gingalewski C, et al. Inflammatory pseudo- ultrastructural, and molecular analysis of 16 cases with a reap- tumor of the alimentary tract: clinical and surgical experience. praisal of clear cell sarcoma-like tumors of the gastrointestinal J Pediatr Surg. 2001;36:169–173. tract. Am J Surg Pathol. 2012;36:857–868. 6 Approach to Hereditary Cancer Syndromes RHONDA K. YANTISS INTRODUCTION ADENOMATOUS POLYPOSES Several inherited disorders are associated with increased Familial Adenomatous Polyposis gastrointestinal cancer risk. Most of these cause epithe- Overview of Molecular Alterations lial gastrointestinal polyposis, although the severity of the polyposis is variable. Others, such as multiple endocrine FAP is an autosomal dominant condition characterized neoplasia type 1 and type 1 neurofibromatosis, are associ- by the presence of hundreds to thousands of adenomatous ated with epithelial, endocrine, and mesenchymal tumors polyps in the colorectum (Figure 6.1). The risk for colorec- of the gastrointestinal tract. Polyposis disorders can be tal cancer approaches 100% by 40 years of age among broadly classified into two groups: adenomatous and patients with this disorder, although less than 1% of hamartomatous polyposes. The former includes entities colorectal carcinomas arise in patients with FAP. Patients such as familial adenomatous polyposis (FAP), MUTYH- with FAP have germline APC mutations, which develop associated polyposis, and Lynch syndrome, whereas at a rate of 1 per 10,000–15,000 live births and result in Peutz–Jeghers syndrome, juvenile polyposis syndrome, an overall disease prevalence of 2.3 to 3.2 per 100,000 and PTEN hamartoma tumor syndrome comprise the lat- individuals. Up to 50% of affected newborns have either ter. Recent advances have provided important insight into de novo germline mutations or parents with germline APC the pathogenesis of all of these disorders and their rela- mosaicism, in which case the mutant allele is limited to tionships to cancer, although application of pathologic, germ cells of the unaffected parent and manifests disease clinical, and molecular criteria still fails to identify all in the offspring. Patients carrying a germline APC muta- patients with heritable cancer syndromes. tion in one copy of the gene develop a “second hit” that The purpose of this chapter is to discuss the clinico- inactivates the other allele and abolishes the tumor sup- pathologic features and differential diagnoses of the most pressor function of cytoplasmic APC. Inactivation of each common heritable cancer syndromes, and provide pathol- allele does not occur as two random independent events. ogists with a framework for the evaluation of these enti- Rather, the nature of the somatic (second) mutation ties. A comprehensive discussion of all tumor syndromes depends on the site of the germline alteration, such that that may have gastrointestinal manifestations is beyond

one allele harbors a mutation near codon 1300. Selection the scope of this chapter, which will focus on epithelial for a mutation in this region ensures that the resultant and hamartomatous polyposis disorders. APC protein retains some functionality such that it may 121 12 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide risk for gastric and small bowel adenocarcinoma (0.5% and 5%, respectively), hepatoblastoma (1.5%), adrenal cortical adenoma, nasopharyngeal angiofibroma, and papillary thyroid carcinoma (2%), particularly the crib- riform morular variant. Congenital hyperpigmentation of the retinal pigmented epithelium is associated with muta- tions between codons 311 and 1444. Pathologic Features and Diagnostic Considerations Patients with FAP develop conventional tubular, villous, and tubulovillous adenomas of the colorectum that are indistinguishable from sporadic adenomas (Figure 6.2). Although untreated patients ultimately develop hundreds to thousands of colorectal adenomas, the increased use of genetic counseling and molecular testing has improved early detection and, as a result, most colectomy specimens now contain far fewer polyps than those removed in the FIGURE 6.1 A resected colon from a patient with familial past (Figure 6.3). These resection specimens should be adenomatous polyposis contains innumerable adenomas carefully evaluated for the number of polyps present in that vary from only a few millimeters to several centimeters order to facilitate classification of the disease and docu- in diameter. Patients with extensive disease, such as this, are at high risk for cancer development. ment the extent of polyposis, particularly when patients have not yet been evaluated with germline testing. In our practice, we entirely submit all polyps spanning at least 1 cm and obtain representative sections of smaller lesions at a rate of one section every 5 to 10 cm of the colon. bind β-catenin. Some patients with milder forms of dis- Any lesions suspicious for invasive adenocarcinoma are ease (ie, attenuated FAP) inherit mutations at the extreme sampled similar to sporadic colorectal carcinomas, and 3′ or 5′ prime ends of APC that lead to partially functional the regional lymph nodes are evaluated. proteins, resulting in fewer polyps and a slightly lower Patients with FAP also develop polyposis of the upper lifetime cancer risk. The molecular features of FAP are gastrointestinal tract. Gastric polyps include adenomas further discussed in Chapter 14. and fundic gland polyps, the latter of which frequently Clinical Features The type of inherited APC mutation dictates the extent and severity of the gastrointestinal polyposis. Although most patients develop innumerable polyps and have a 100% risk of colorectal cancer if not treated by prophy- lactic proctocolectomy, those with attenuated FAP gener- ally develop fewer than 100 colorectal polyps and have a slightly lower lifetime colorectal cancer risk of approxi- mately 80%. Extraintestinal manifestations are also related to the nature of the underlying germline mutations. Several named variants describe specific combinations of pheno- typic characteristics. Gardner syndrome denotes the con- stellation of osteomas, mesenteric fibromatosis, cutaneous cysts, lipomas, and dental abnormalities that occur in addition to polyposis. This form of disease is commonly associated with germline APC mutations in codons 1395 to 1493. Patients with Crail syndrome have gastrointes- FIGURE 6.2 Cross sections through the colonic mucosa tinal polyposis in combination with medulloblastomas, of patients with familial adenomatous polyposis can reveal ependymomas, or astrocytomas. Extracolonic manifesta- single dysplastic crypts (arrow) that represent the earliest tions of the disease are numerous and include increased identifiable histologic stage of adenoma development. 6 Approach to Hereditary Cancer Syndromes 12 3 FIGURE 6.3 Patients with familial adenomatous polyposis may come to clinical attention before the entire colonic mucosa is carpeted by adenomas. This patient already has several tiny polyps that stud the mucosa, as well as three synchronous cancers (arrows). show dysplasia (Figure 6.4). Indeed, approximately 50% FIGURE 6.5 More than half of the patients with FAP of patients with FAP have dysplasia in some of their fun- and fundic gland polyps have dysplasia in some of dic gland polyps. The dysplasia in these cases is typically the fundic gland polyps. The dysplasia is usually low- low-grade and focal, although occasional polyps show grade with enlarged, hyperchromatic nuclei and nuclear more extensive abnormalities or high-grade dysplasia pseudostratification. (Figure 6.5). The biologic risk of this finding is quite low and, thus, patients can be safely monitored with surveil- lance endoscopy and regular removal of large lesions without prophylactic gastrectomy. Nonsyndromic fundic gland polyps show dysplasia much less frequently (less than 5%) than those associated with FAP. Thus, patholo- gists may suggest the possibility of a polyposis disorder when fundic gland polyps with dysplasia are encoun- tered, particularly if the patient has multiple gastric polyps. Adenomas of the small bowel are more common among patients with APC mutations between codons 976 and 1067. They show a predilection for the ampulla and nonampullary duodenum as well as the proximal jejunum. Development of nonampullary adenomas of the duode- num is extremely uncommon in the sporadic setting and, in fact, the pathologist should raise the possibility of FAP when these lesions are encountered, particularly if they are multiple. Differential Diagnosis The diagnosis of FAP is generally straightforward when patients have typical disease manifestations. Other dis- FIGURE 6.4 Patients with familial adenomatous orders associated with gastrointestinal polyps can simu- polyposis commonly develop multiple fundic gland polyps. late the endoscopic appearance of FAP, however. These This patient has numerous sessile nodules in the gastric include lymphomatoid polyposis, inflammatory bowel dis- body and fundus. ease with pseudopolyps, and hamartomatous polyposes, 12 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide although all of these entities are readily distinguishable on gastric adenomas. Other associated malignancies include biopsy analysis. The differential diagnosis of multiple gas- carcinomas of skin, breast, ovary, and urinary bladder. trointestinal adenomas is more problematic. Attenuated Some patients develop sebaceous tumors in combination FAP and MUTYH-associated polyposis manifest with with the polyposis, thereby mimicking the features of the similar numbers of colorectal adenomas that are histologi- Muir–Torre variant of Lynch syndrome. cally indistinguishable, and require germline analysis for diagnosis. However, patients with MUTYH-associated Pathologic Features and Diagnostic Considerations polyposis may present with nondysplastic and dysplastic serrated polyps, both of which are generally lacking in MUTYH-associated polyposis was first described in 2002 patients with FAP. Some patients with Lynch syndrome and, thus, the full spectrum of phenotypic and molecular have multiple colorectal adenomas, as well as adenomas alterations has not been elucidated. Most patients have of the small bowel and periampullary region. One may between 10 and 100 colorectal polyps, although rare suspect Lynch syndrome if adenomas contain increased individuals have several hundred polyps and some pres- intraepithelial lymphocytes or show high-grade features ent with colorectal carcinoma in the absence of other despite relatively small size. Immunohistochemical stains lesions. Most adenomas are conventional intestinal-type for DNA mismatch repair proteins may show loss of stain- adenomas with low-grade dysplasia that simulate FAP, ing typical of Lynch syndrome when adenomas are large, but hyperplastic polyps and sessile serrated adenomas also located in the proximal colon, or show high-grade dys- occur. Furthermore, MUTYH mutations may underlie the plasia. Carcinomas that develop in patients with Lynch development of serrated (hyperplastic) polyposis in some syndrome show morphologic heterogeneity, tumor infil- cases. Carcinomas associated with MUTYH-associated trating lymphocytes, and a Crohn-like lymphoid response. polyposis show a predilection for the proximal colon. MUTYH-Associated Polyposis Differential Diagnosis Overview of Molecular Alterations The differential diagnosis of MUTYH-associated polypo- sis includes attenuated FAP, Lynch syndrome, and serrated Some patients with gastrointestinal polyposis have an polyposis. Although germline mutational testing conclu- autosomal recessive disease that results from biallelic sively distinguishes between these entities, other clinico- inactivating mutations in the base excision repair gene, pathologic features can be helpful. Attenuated FAP is not MUTYH, on chromosome 1p32-34. Patients with these associated with increased numbers of serrated polyps, inactivating MUTYH mutations have a predilection for whereas this finding is commonly present in MUTYH- acquiring somatic APC mutations and, as a result, develop associated polyposis. The latter has not been reported in numerous colorectal polyps that simulate the phenotype combination with tumors of the central nervous system, of attenuated FAP. Patients can also develop transversion mesenteric fibromatosis, or other extraintestinal manifes- mutations or spontaneous promoter methylation affecting tations of Gardner syndrome. Both MUTYH-associated MLH1, so resultant tumors may have either chromosomal polyposis and Lynch syndrome may be associated with or microsatellite instability (MSI). Patients with polypo- the development of MSI-H colorectal carcinomas. Thus sis due to MUTYH mutations have a lifetime colorectal far, those associated with MUTYH-associated polyposis cancer risk of approximately 80%. Recommended sur- show MLH1 deficiencies, either in the form of promoter veillance mirrors that of patients with attenuated FAP; methylation or mutations. patients should undergo complete biannual colonoscopy beginning at 18 to 20 years of age and upper endoscopic examination beginning at age 25 to 30 years. Lynch Syndrome (Hereditary Non-Polyposis Colon Cancer) Clinical Features Overview of Molecular Alterations The incidence of MUTYH-associated polyposis is approx- Lynch syndrome is an autosomal dominant disorder char- imately 1 in 10,000 persons based on the 2% estimated acterized by early-onset colonic carcinoma due to inac- prevalence of heterozygous mutations in Caucasian tivating germline mutations in mismatch repair genes. populations. This disorder accounts for less than 1% of These genes (MLH1, MSH2, MSH6, and PMS2) encode colorectal carcinomas, but approximately one-third of proteins that form complexes and patrol the genome to patients with colorectal polyposis and wild-type APC have correct DNA mismatches. Microsatellites are prone to this syndrome. Nearly 20% of patients with MUTYH- mismatch repair errors owing to the tendency for DNA associated polyposis develop duodenal adenomas. These polymerase to slip over repetitive sequences in these patients have a 4% lifetime risk of duodenal carcinoma, areas. Failure of the mismatch repair system to correct and a substantial number have fundic gland polyps and/or errors results in their propagation, such that subsequent 6 Approach to Hereditary Cancer Syndromes 12 5 microsatellite sequences show expansion or contraction of PMS2 or MSH6. These patients with constitutional mis- length in tumor DNA compared to that of non-neoplastic match repair deficiency develop Lynch-syndrome-related tissues from the same patient. cancers in late adolescence or early adulthood and often Most laboratories now use quasimonomorphic have multiple colorectal polyps, thereby mimicking FAP. mononucleotide markers (BAT25, BAT26, NR-1, NR-24, Extracolonic manifestations of constitutional mismatch MONO-27) to assess for MSI. Tumors with instability repair deficiency include cafè au lait spots, leukemia or at two or more markers are classified as showing MSI- lymphoma, gliomas, and extracolonic gastrointestinal H, whereas those without MSI at any marker are classi- malignancies. fied as microsatellite stable and those with MSI at one marker are considered to be indeterminate. Other rare Pathologic Features and Diagnostic Considerations causes of Lynch syndrome include heritable epigenetic inactivation of MLH1 through MLH1 germline pro- Colorectal carcinomas with MSI-H characteristically moter methylation, and a germline deletion affecting occur in the proximal colon, where they form large, TACSTD1 (EPCAM) that eliminates the TACSTD1 bulky masses (Figure 6.6). Tumors tend to have a circum- stop codon, leading to the formation of EPCAM–MSH2 scribed, rather than infiltrative, invasive front and show fusion transcripts and MSH2 inactivation. The molecular histologic heterogeneity, which may include combinations mechanisms underlying MSI are discussed more fully in of conventional carcinoma admixed with areas of med- Chapter 14. Immunohistochemical stains for mismatch ullary growth, mucinous differentiation, or signet ring repair proteins may be used as a surrogate marker of cells (Figure 6.7A–C). Approximately 50% of mucinous MSI, with the added benefit of identifying the deficient colorectal carcinomas are MSI-H, whereas nearly 90% gene based on the pattern of staining (see Table 6.1), as of medullary carcinomas of the colon are MSI-H. These discussed further in Chapter 13. cancers are often associated with tumor infiltrating lym- phocytes (Figure 6.7D) or the presence of lymphoid aggre- gates at the periphery of the tumor (Figure 6.7E). Both Clinical Features intraepithelial lymphocytes and the Crohn-like lymphoid Lynch syndrome accounts for 2% to 4% of all colorectal response likely represent a host immune response to neo- carcinomas, and affected patients have an 80% lifetime antigens elaborated as a result of MSI-H. risk of colorectal carcinoma. Most tumors develop approx- Patients with Lynch syndrome can sometimes initially imately 20 years earlier than they do in the general popu- present with extracolonic malignancies, in which case the lation (mean age at onset: 44 years), so affected patients diagnosis is rarely a clinical consideration.

Gastric and undergo screening colonoscopy at age 20 to 25 years with surveillance examinations every 1 to 2 years thereafter. Lynch syndrome is also associated with an increased risk for extracolonic cancers of the endometrium, ovary, renal pelvis and ureter, small intestine, stomach, and hepatobi- liary tract. Muir–Torre syndrome and Turcot syndrome represent named variants of Lynch syndrome. Muir–Torre syndrome features internal malignancies associated with sebaceous neoplasms, whereas patients with Turcot syn- drome have glioblastoma multiforme in combination with intestinal neoplasms. Rare patients have germline mutations affecting two mismatch repair genes, either in the form of homozygous biallelic loss or compound heterozygosity usually affecting TABLE 6.1 Interpretation of Mismatch Repair Protein Immunohistochemistry Immunostaining Microsatellite Profile Defective Gene Status FIGURE 6.6 A 59-year-old male underwent colonoscopy Loss of MLH1 and PMS2 MLH1 MSI-H after presenting with occult blood loss. He was found Loss of MSH2 and MSH6 MSH2 or TACSTD1 MSI-H to have a nearly obstructing, fungating mass in the (EPCAM) transverse colon. Biopsies revealed a mucinous Loss of PMS2 PMS2 MSI-H carcinoma that proved to be MLH1 deficient due to a Loss of MSH6 MSH6 MSI-H or MSS germline mutation. 12 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 6.7 Microsatellite unstable carcinomas may display a variety of growth patterns, often in combination. Medullary carcinomas are composed of tumor cells arranged in trabeculae without overt gland formation (A). Mucinous carcinomas contain lace-like arrangements of neoplastic epithelial cells floating within pools of mucin (B). Some tumors have a conventional appearance with fused or cribriform glands (C). Intraepithelial lymphocytes are characteristic of tumors with MSI-H, regardless of the morphologic appearance of the lesion (D), and lymphoid aggregates at the periphery of the tumor may also be seen (E). small intestinal adenocarcinomas associated with Lynch of APC or CTNNB1 mutations and develop MSI-H late syndrome frequently show morphologic heterogeneity in their evolution. These features have important clinical with high-grade areas or foci of mucinous differentiation. implications. Patients or clinicians may ask pathologists Tumor infiltrating lymphocytes are variably present, but to evaluate adenomas for possible Lynch syndrome when when detected, should prompt pathologists to consider the multiple colorectal polyps are present, but this may not possibility of MSI-H and Lynch syndrome. yield meaningful results. For example, Lynch syndrome The precursor lesion of Lynch-syndrome-associated is unlikely to be present in patients with multiple serrated carcinoma is a conventional adenoma, not a serrated neo- polyps. Nondysplastic serrated polyps do not show MSI-H plasm. Adenomas of affected patients show inactivation and dysplastic serrated polyps with MSI-H develop via 6 Approach to Hereditary Cancer Syndromes 127 MLH1 promoter methylation, not germline mutations. hypermethylation and show loss of immunostaining for Additionally, assessment of adenomas for Lynch syn- MLH1 and PMS2, similar to MLH1 deficient carcinomas drome often yields unreliable results, even among patients of Lynch syndrome. In fact, sporadic MSI-H carcinomas who carry a mutation. MSI develops late in adenoma pro- are indistinguishable from Lynch-related MLH1 deficient gression and, thus, failure to detect mismatch deficiency tumors based on polymerase chain reaction (PCR) for in an adenoma does not necessarily exclude the possibility MSI and immunohistochemistry alone. However, approx- of Lynch syndrome in a given patient. Most data suggest imately 50% of sporadic MSI-H cancers also show BRAF that screening adenomas among unselected young patients V600E mutations, whereas this abnormality is not typical (less than 40 years of age) has a very low yield for detect- of MLH1-deficient tumors in Lynch syndrome. Mutations ing Lynch patients owing to the relatively high prevalence in BRAF may be detected in extracted tumoral DNA of sporadic adenomas in this patient population. For or with recently available immunohistochemical stains these reasons, universal testing of adenomas for Lynch directed against the mutant protein. The molecular and syndrome is not recommended. However, it is reasonable immunohistochemical diagnosis of Lynch syndrome is to assess large, or proximally located, adenomas for mis- further discussed in Chapters 11, 13, and 14. match repair protein expression when there is a clinical suspicion for a heritable cancer syndrome. HAMARTOMATOUS POLYPOSES Differential Diagnosis Peutz–Jeghers Syndrome The differential diagnosis of Lynch syndrome includes attenuated FAP and MUTYH-associated polyposis when Overview of Molecular Features patients have colorectal cancer in combination with Peutz–Jeghers syndrome is an autosomal dominant hered- colonic adenomas. The distinction is straightforward in itary hamartomatous polyposis syndrome characterized most cases, as cancers associated with Lynch syndrome by generalized gastrointestinal polyposis in combina- show typical morphologic features as well as immunohis- tion with a variety of extraintestinal manifestations (see tochemical loss of one or more DNA mismatch repair pro- Table 6.2). Approximately 70% of patients have detectable teins. The more problematic differential diagnosis lies with mutations in LKB1 (STK11), which is located on chromo- sporadic MSI-H colorectal cancers. Most sporadic MSI-H some 19p13.3. This tumor suppressor gene encodes ser- colorectal cancers develop as a result of MLH1 promoter ine/threonine kinase 11, alternatively termed liver kinase TABLE 6.2 Classic Features of Hamartomatous Polyposis Syndromes of the Gastrointestinal Tract Peutz–Jeghers Syndrome Juvenile Polyposis Syndrome PTEN Hamartoma Tumor Syndrome Mutant gene LKB1 (STK11) SMAD4 PTEN BMBR1A SDHB or SDHD KLLN PIK3CA AKT1 Gene function Serine threonine kinase regulates TGF-β mediated signal Tumor suppressor gene adenine monophosphate activated transduction protein kinase (AMPK) Mucocutaneous Perioral, buccal, and conjunctival Oral papillomas, tricholemmomas, manifestations pigmentation; perioral pigment acral keratoses fades with age, but buccal and conjunctival lesions persist Other abnormalities Ovarian tumors (sex cord–stromal Cranial and cardiac abnormalities Thyroid disease: autoimmune tumor with annular tubules) Cleft palate thyroiditis and carcinoma Cervical cancer (adenoma malignum) Polydactyly Breast disease: fibrocystic breast Testicular tumors (large cell calcifying Intestinal malrotation disease and carcinoma Sertoli cell tumor) Hereditary hemorrhagic Macrocephaly Mammary carcinoma telangiectasia Mental impairment Pancreatic ductal adenocarcinoma Vascular malformations Glycogenic acanthosis of esophagus Hypertrophic osteoarthropathy Gastrointestinal polyp Small intestine most affected, Colon most affected, followed by Colon most affected, followed by location followed by colon and stomach stomach and small bowel stomach and small bowel Gastrointestinal Colorectum most affected, followed Colorectum most affected, Colorectum most affected, followed by carcinomas by stomach and small bowel followed by stomach and small stomach bowel 12 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide B1, which normally maintains cell polarity and inhibits those of adenomas. Patients with Peutz–Jeghers syndrome cell growth by activating other kinases and regulating are at markedly increased risk for gastrointestinal and the activity of adenine monophosphate activated protein extraintestinal cancers; more than 90% of the patients kinase (AMPK). Phosphorylation of AMPK by STK11 will develop some type of malignancy by 65 years of age. leads to its activation, which is important to cell metabo- The lifetime risk of colorectal cancer is 35% to 40%, fol- lism and homeostasis. Loss of STK11 results in cellular lowed in decreasing frequency by pancreatic cancer (35%), disorganization and facilitates tumor growth. The disease gastric cancer (28%), and small bowel cancer (10%–15%). phenotype tends to be more severe in patients with trun- Children and young adults usually present with abdomi- cating mutations compared to those who have missense nal pain or bleeding, whereas malignant complications are mutations. more common among older adults. Clinical Features Pathologic Features and Diagnostic Considerations Peutz–Jeghers syndrome affects one in 200,000 persons in Solitary hamartomatous polyps that resemble Peutz– the United States and is characterized by gastrointestinal Jeghers polyps have been described as sporadic lesions in hamartomatous polyps, mucocutaneous pigmentation, the older literature. However, increased use of molecular and an increased risk of malignancy affecting multiple testing and improved clinical assessment have shown that organ systems. Clinical criteria for the diagnosis include virtually all solitary polyps with features of Peutz–Jeghers (a) at least three Peutz–Jeghers polyps; (b) any number polyps occur in patients with germline STK11 muta- of Peutz–Jeghers polyps in a patient with afflicted fam- tions or those with clinical features suggestive of Peutz– ily members; (c) mucocutaneous pigmentation in a patient Jeghers syndrome. Thus, solitary Peutz–Jeghers polyps with afflicted family members; or (d) any number of should be considered a forme fruste of the syndrome until Peutz–Jeghers polyps occurring in a patient with mucocu- proven otherwise, and prompt evaluation for a heritable taneous pigmentation. syndrome. Peutz–Jeghers polyps are most numerous in the small Peutz–Jeghers polyps contain lobules of nondysplastic intestine, where they are prone to cause intermittent intus- epithelium and associated lamina propria in the submu- susception. Virtually all patients with hamartomas of the cosa, and up to 10% of small bowel polyps are associated colon, stomach, and appendix also have involvement of with aggregates of mucosa distributed in all layers of the the small bowel. Polyps are round with smooth surfaces wall (Figure 6.9A–E). These mucosal elements are sur- that may be eroded or, more commonly, resemble the rounded by prominent bundles of smooth muscle cells that background mucosa (Figure 6.8A–B). Pedunculated pol- display a pronounced, arborizing pattern, particularly in yps tend to have thick stalks that may be much longer than polyps of the small intestine. Slightly more than 10% of (A) (B) FIGURE 6.8 A gastric Peutz–Jeghers polyp forms an irregular, lobulated excrescence on a mucosal fold (A). The same patient had a multinodular, nearly obstructing hamartoma in the fourth part of the duodenum (B). 6 Approach to Hereditary Cancer Syndromes 12 9 (A) (B) (C) (D) (E) (F) FIGURE 6.9 Peutz–Jeghers polyps develop throughout the gastrointestinal tract. Characteristic Peutz–Jeghers polyps, as seen here in the small bowel, contain lobules of mucosal elements surrounded by bundles of arborizing smooth muscle (A–C). However, these features are less well-developed in gastric polyps (D). This colonic Peutz–Jeghers polyp contains lobules of non-neoplastic mucosal elements surrounded by bundles of smooth muscle cells (E). Some lesions contain areas of dysplasia similar to that seen in tubular or villous adenomas (F). Peutz–Jeghers polyps show dysplasia, which is usually polyp of the small intestine is straightforward, whereas low-grade and more commonly encountered in lesions of gastric and colonic polyps are more problematic because the colorectum (Figure 6.9F). Carcinomas may develop in they lack well-developed arborizing bundles of smooth either the polypoid or nonpolypoid mucosa and resemble muscle cells. Gastric Peutz–Jeghers polyps have a lobular sporadic colorectal carcinomas. architecture and usually lack the cystically dilated glands and pits typical of juvenile polyps and hyperplastic pol- yps. They also contain normal-appearing lamina propria, Differential Diagnosis rather than the edematous, inflamed stroma of hyperplas- Peutz–Jeghers polyps most commonly simulate other types tic polyps and juvenile polyps. Colonic Peutz–Jeghers pol- of hamartomatous polyps. The diagnosis of a Peutz–Jeghers yps contain rounded aggregates of epithelium and lamina 13 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide propria without prominent crypt dilation, which is more to the splenic flexure. Signs and symptoms usually reflect typical of juvenile polyps, and there can be substantial gastrointestinal bleeding, and include heme-positive morphologic overlap that causes diagnostic confusion. stools, anemia, and passage of blood or tissue (autoampu- Stromal edema with inflammation and mucosal cysts are tated polyps) per rectum. sometimes present in Peutz–Jeghers polyps of the colon to Juvenile polyposis syndrome affects one in 100,000 such an extent that they may not be discernible from juve- persons in the United States and shows variably severe dis- nile polyps based on histologic evaluation alone. ease depending on the type of mutation present. Infantile Peutz–Jeghers polyps can also simulate the features juvenile polyposis presents within the first two years of of mucosal prolapse polyps, adenomas with misplaced life in patients without a family history of juvenile pol- epithelium, and invasive adenocarcinomas. Mucosal pro- yposis syndrome. Manifestations include generalized lapse polyps contain prominent bundles of smooth muscle gastrointestinal polyposis with bleeding, malabsorptive cells emanating from the muscularis mucosae, but also diarrhea, and protein-losing enteropathy, which can be show other distinguishing features, such as erosions, life-threatening. Hamartomatous polyps are limited to the crypt regeneration, and hyperplasia, ischemic injury, colons of patients with juvenile polyposis coli. Generalized and inflammation. Lobules of mucosal elements in the juvenile polyposis manifests with numerous gastrointes- bowel wall simulate epithelial misplacement in adenomas, tinal polyps of the stomach, small intestine, and colon. although Peutz–Jeghers polyps contain non-neoplastic epi- Diagnostic criteria include (a) three or more colorectal thelium and lack hemosiderin deposits, extruded mucin, juvenile polyps, (b) any number of extracolonic juvenile hemorrhage, fibrosis, and other features of trauma. polyps, or (c) any number of juvenile polyps in patients Hamartomatous elements in the bowel wall may simulate with a family history of the

syndrome. invasive adenocarcinoma, particularly when they contain Extraintestinal congenital abnormalities can be found dysplastic epithelium. Helpful diagnostic features include in 10% to 20% of patients with juvenile polyposis syn- the lobular arrangement of non-neoplastic, or low-grade drome. Some patients with SMAD4 mutations develop dysplastic, and presence of epithelium invested with lam- hereditary hemorrhagic telangiectasia characterized by ina propria, rather than the desmoplastic stroma of an systemic vascular malformations, and pulmonary arte- invasive carcinoma. riovenous malformations as well as hypertrophic osteo- arthropathy. Although patients with juvenile polyposis Juvenile Polyposis Syndrome syndrome have gastrointestinal hamartomas, they are at increased risk for carcinomas of the colorectum (cumula- Overview of Molecular Features tive risk of nearly 70% by 60 years of age), stomach, pan- Juvenile polyposis syndrome is an autosomal dominant creas, and proximal small intestine. hereditary hamartomatous polyposis syndrome result- Patients with juvenile polyposis syndrome develop ing from defects in the signal transduction pathway initi- numerous colorectal polyps, ranging from a few to several ated by TGF-β. Approximately 20% of patients prove to hundred lesions (Figure 6.10A–C). Small intestinal and have germline mutations in SMAD4, located on chromo- gastric polyps also occur in patients with generalized juve- some 18q21.1, and 20% to 25% of patients have muta- nile polyposis. Small lesions are sessile and erythematous tions in BMBR1A, located on chromosome 10q22.3. As polyps that resemble hyperplastic polyps, whereas larger is the case in other hereditary polyposis disorders, specific polyps are usually pedunculated with erosions or ulcers mutations give rise to phenotypically different patterns (Figure 6.10D). Some juvenile polyps have a mulberry-like of disease. Polyposis of the upper gastrointestinal tract is appearance and these have been termed “atypical juvenile more common among patients with SMAD4 mutations, polyps.” Atypical juvenile polyps occur only in associa- whereas cardiac defects are more common among patients tion with juvenile polyposis syndrome. with germline BMPR1A mutations. Large deletions that encompass BMBR1A and PTEN, two contiguous tumor Pathologic Features and Diagnostic Considerations suppressor genes, have been described in infantile juvenile polyposis. Of note, less than 50% of patients with juvenile Juvenile polyps are round, smooth lesions with surface polyposis syndrome have a family history of the disease. erythema reflecting the presence of inflamed, eosinophil- rich stroma and frequent erosions (Figure 6.11A–B). Cross Clinical Features sections reveal numerous cysts that correspond to dilated crypts (colon) or glands (stomach) in the polyp head. Some Sporadic juvenile polyps are relatively common, affecting polyps also contain clusters of ganglion cells in the mucosa. 1% to 2% of pediatric patients. They are smooth, some- Sporadic and syndromic juvenile polyps can be indistin- what friable mucosal-based polyps that may be peduncu- guishable, although many syndromic polyps display less lated or sessile. Most sporadic juvenile polyps develop in crypt dilation and stromal edema. Atypical juvenile pol- the rectum, though they infrequently develop proximal yps occur exclusively in association with the syndrome 6 Approach to Hereditary Cancer Syndromes 131 (A) (B) (C) (D) FIGURE 6.10 A 3-year-old girl presented with anal bleeding and underwent colonoscopy. She was found to have five polyps, including three in the rectum and two in the abdominal colon, all of which proved to be juvenile polyps. All were smooth, sessile polyps with surface erythema and erosions (A). A 4-year-old girl was evaluated for hematochezia and proved to have juvenile polyposis coli. Multiple large polyps were present in the rectosigmoid colon, with stigmata of recent bleeding (B). The same patient had an irregular, pedunculated polyp in the sigmoid colon (C). Another patient with juvenile polyposis syndrome underwent a gastric resection. Juvenile polyps carpet the mucosa and simulate the appearance of Ménétrier disease (D). and usually develop in the colon (Figure 6.11C). They con- the appearance of Peutz–Jeghers polyps, although juvenile tain crowded, irregularly shaped crypts with little inter- polyps with this finding also typically contain cystically vening stroma. Some atypical juvenile polyps display areas dilated crypts and inflamed stroma, both of which are of dysplasia, which is usually low-grade (Figure 6.11D). less frequent in Peutz–Jeghers polyps. Distinction between Carcinomas can develop in either polypoid or nonpolyp- juvenile polyposis syndrome and inflammatory-type pol- oid mucosa. yps due to other conditions is more problematic. Colonic juvenile polyps may be histologically indistinguish- able from polyps seen in inflammatory bowel disease, Differential Diagnosis inflammatory “cap” polyposis, and Cronkhite–Canada Juvenile polyposis syndrome simulates other hamartoma- syndrome, whereas gastric involvement can mimic hyper- tous syndromes and inflammatory conditions. Fascicles plastic polyps or Ménétrier disease, particularly in biopsy of smooth muscle cells may be prominent and simulate samples. All of these disorders display inflammatory 13 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 6.11 Juvenile polyps contain cystically dilated crypts that are irregularly dispersed in inflamed stroma (A), which contains a mixed inflammatory cell infiltrate rich in eosinophils (B). Atypical juvenile polyps display crowded crypts with less conspicuous cysts and stroma, thereby more closely resembling inflammatory polyps that develop in other conditions, such as inflammatory bowel disease (C). Atypical juvenile polyps may contain areas of dysplasia, which is often low-grade and similar to that seen in tubular or villous adenomas (D). changes in the nonpolypoid mucosa, which is normal in result of germline PTEN mutations. This gene encodes patients with juvenile polyposis syndrome. phosphatase and tensin homolog, which is a 403 amino acid phosphatase with tumor suppressor activity. The PTEN Hamartoma Tumor Syndrome PTEN protein plays key regulatory roles in cell cycling and migration, apoptosis, and genomic stability. Patients Overview of Molecular Features who have phenotypic manifestations of disease but lack PTEN hamartoma tumor syndrome is an autosomal PTEN mutations may harbor other germline alterations, dominant cancer syndrome that encompasses the pre- including mutations in succinyl dehydrogenase complex viously named entities Cowden syndrome, Lhermitte– subunits B (SDHB) on 1p35-36 or D (SDHD) on 11q23, Duclos syndrome, and Bannayan–Ruvalcaba–Riley hypermethylation of the KLLN promoter, and PIK3CA syndrome, as well as related disorders that develop as a or AKT1 mutations. 6 Approach to Hereditary Cancer Syndromes 13 3 Clinical and Endoscopic Features juvenile polyps (Figure 6.12B–C). Some PTEN hamar- tomatous polyps contain bundles of myofibroblastic PTEN hamartoma tumor syndrome affects approxi- spindle cells (Figure 6.12D), ganglion cells, or fat in the mately one in 200,000 persons. Between 35% and 40% polyp head, all of which may be helpful diagnostic fea- of patients develop numerous hamartomatous polyps tures. Lipomas, ganglioneuromas, and lymphoid nodules throughout the gastrointestinal tract in combination with are often detected as small, incidental lesions upon colo- a variety of extraintestinal abnormalities. Gastrointestinal noscopic examination. Indeed, one should consider the manifestations include glycogenic acanthosis, hyperplas- possibility of PTEN hamartoma tumor syndrome in any tic and inflammatory polyps, adenomas, and mesenchy- patient with multiple mesenchymal lesions or “hyper- mal polyps (Figure 6.12). Breast and thyroid disease are plastic” polyps that contain fat or spindle cells in the the most common extraintestinal abnormalities. Female lamina propria. patients develop fibrocystic breast disease and breast can- cer at a much higher rate and at an earlier age than women in the general population (estimated lifetime cancer risk Differential Diagnosis of 25% to 50% compared to 10%). Hashimoto thyroid- itis is relatively common and the lifetime risk for thyroid Diffuse esophageal glycogenic acanthosis is virtually cancer is nearly 10%. Other extraintestinal manifestations pathognomonic of PTEN hamartoma tumor syndrome, include oral papillomas, cutaneous trichilemmomas, and and essentially has no histologic differential diagnosis, acral keratoses, macrocephaly, and mental impairment. although the endoscopic appearance may simulate either The above-mentioned features are characteristic of eosinophilic esophagitis or Candida infection. Syndromic Cowden syndrome, although several phenotypic variants hamartomatous polyps with prominent spindle cells can exist. Patients with Lhermitte–Duclos disease have some be indistinguishable from sporadic leiomyomas, benign of these findings in addition to dysplastic gangliocytomas stromal/epithelial polyps (ie, perineurioma), and ganglio- of the cerebellum. Patients with Bannayan–Ruvalcaba– neuromas, but the presence of multiple lesions is strongly Riley syndrome develop hamartomas in combination with suggestive of PTEN hamartoma tumor syndrome. Other lipomatosis, hemagiomas, and mixed vascular and fatty gastrointestinal hamartomas that develop in association tumors involving skin and viscera. Additional craniofacial with PTEN hamartoma tumor syndrome bear a resem- features include downslanting palpebral fissures, ocular blance to inflammatory-type polyps and display variably hypertelorism, pseudopapilledema and prominent corneal dilated crypts or pits enmeshed in inflamed lamina pro- nerves, café au lait spots, acanthosis nigricans, and wart- pria, thereby simulating juvenile polyposis and Cronkhite– like lesions. Most affected males have pigmented macules Canada syndrome, as well as inflammatory-type polyps on the shaft and glans penis. Musculoskeletal abnormali- unassociated with polyposis syndromes. Isolated gas- ties are present in more than 50% of patients and usually tric hyperplastic polyps and mucosal abnormalities of reflect hypotonia of proximal muscles due to abnormal Ménétrier disease simulate the features of gastric ham- lipid storage, although hyperextensible joints, scoliosis, artomas, whereas the differential diagnosis of colorectal and pectus excavatum are common. Autism spectrum dis- lesions includes inflammatory-type polyps associated with order with macrocephaly, Proteus syndrome, and VATER mucosal prolapse, inflammatory cap polyposis, or inflam- with macrocephaly have also been reported in association matory bowel disease. with PTEN mutations. The distribution of disease in the gastrointestinal tract aids distinction of PTEN hamartoma tumor syn- drome from inflammatory conditions that may be con- Pathologic Features sidered in the differential diagnosis. The intervening Diffuse glycogenic acanthosis of the esophagus devel- nonpolypoid mucosa of patients with PTEN hamartoma ops in 20% to 80% of affected patients (Figure 6.12A). tumor syndrome is essentially normal, whereas many The endoscopic features of gastrointestinal polyps are of the entities in the differential diagnosis (inflamma- variable, depending on the relative amounts of epithe- tory bowel disease or Cronkhite–Canada syndrome, lium, stroma, and inflammation present in the lesions. for example) have inflamed intervening nonpolypoid Adenomas may appear as sessile or pedunculated mucosa. The PTEN hamartoma tumor syndrome should mucosa-based polyps similar to sporadic polyps, whereas be considered in any patient with multiple inflammatory- mesenchymal lesions are dome-shaped, smooth polyps type polyps, especially if they occur in combination with surfaced by normal mucosa and those with inflamma- adenomas and mesenchymal polyps, or contain a myo- tory changes display erythema or erosions. Gastric fibroblastic spindle cell proliferation in the lamina pro- lesions mimic hyperplastic polyps and contain cystically pria. However, many PTEN hamartomas lack distinctive dilated pits lined by mucin-depleted or hypermucinous features, so evaluation of patients with multiple juvenile epithelium. Intestinal lesions contain cystic crypts within or inflammatory-type polyps should include molecular an expanded and inflamed lamina propria similar to testing for this possibility. 13 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 6.12 A 25-year-old woman underwent surveillance endoscopy for Cowden syndrome. Glycogenic acanthosis appears as numerous white plaques throughout the esophagus (A). Numerous hamartomas form smooth, round nodules in the duodenum (B). Colonic hamartomas appear as subtle excrescences surfaced by normal mucosa (C). This hamartomatous colon polyp consists of normal-appearing epithelium and a spindle-cell-rich cellular proliferation in the lamina propria (D). SUMMARY AND CONCLUSIONS and intraepithelial lymphocytes. However, the onset of MSI-H is a relatively late event in Lynch-related neopla- Pathologists play a major role in guiding patient man- sia, so its absence from an adenoma (or preservation of agement and endoscopic surveillance of patients with mismatch repair protein staining) does not necessarily suspected heritable cancer syndromes. Many affected exclude the possibility of Lynch syndrome. patients do not have known genetic alterations at the time Histologically similar hamartomatous polyps can of initial evaluation, and disease manifestations are often occur in patients with different types of syndromic pol- variable. Careful pathologic assessment of morphologic yposis (see also Table 6.2). Key features in the past medi- features and distribution of disease is vital to the evalua- cal history should alert pathologists to the possibility of tion of patients with multiple gastrointestinal polyps. The a hamartomatous disorder. These include the presence of distinction between subtypes of adenomatous polyposis hyperplastic and inflammatory-type polyps, particularly coli is best made through germline mutational testing, if they are multiple, affect the upper and lower gastro- although some histologic clues are typical of lesions asso- intestinal tract, or develop in the proximal small bowel ciated with Lynch syndrome, such as tumor heterogeneity where such lesions are uncommon. Multiple diagnoses of 6 Approach to Hereditary Cancer Syndromes 13 5 ganglioneuroma, perineurioma, and lipoma in biopsies a flat lesion in the right colon revealed a sessile serrated from a

single patient should also arouse suspicion, par- polyp (Figure 6.13B). There was no known cancer history ticularly if they occur in combination with hyperplastic in the patient’s parents or grandparents. An upper endos- polyps or adenomas. copy was negative. The patient underwent a total proc- tocolectomy for what was deemed to be a polyposis not amenable to endoscopic management. SPECIFIC ILLUSTRATIVE EXAMPLES Gross examination of the colectomy specimen revealed 85 to 95 polyps, ranging in size from 0.1 to Case 1 3 cm (Figure 6.13C), which were right-side predominant (Figure 6.13D). All of the polyps greater than 1 cm in A 45-year-old man with no significant past medical history size were entirely submitted; 15 additional polyps from presented to his primary care physician with rectal bleed- throughout the colon were sampled in five cassettes. The ing and abdominal pain. Colonoscopy demonstrated an larger polyps represented adenomas, while several ses- extensive polyposis, with lesions ranging from a few mil- sile serrated polyps and hyperplastic polyps were also limeters to 3 cm. Snare polypectomy of one of the larger noted. lesions located in the distal transverse colon revealed a The patient was referred to a genetic counselor and tubulovillous adenoma (Figure 6.13A), while biopsy of APC mutation testing was pursued, which failed to (A) (B) (C) (D) FIGURE 6.13 A 45-year-old man with MUTYH-associated polyposis had an attenuated polyposis characterized by a combination of adenomas (A) and serrated polyps (B). The colectomy specimen contained fewer than 100 polyps, ranging in size from 0.1 to 3 cm (C). The polyps were right-side predominant (D). 13 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide demonstrate a germline mutation. This was followed by Case 2 MUTYH mutation testing, and the patient was found to be a Y179C/G396D compound heterozygote. Genetic testing A 40-year-old man presented to the emergency depart- was offered to the patient’s four siblings, each of whom ment in a rural hospital with upper gastrointestinal bleed- was found to have a 25% risk of being affected. ing. He had no significant past medical history and no Because MUTYH-associated polyposis was fairly previous episodes of bleeding or gastrointestinal symp- recently described, the full spectrum of phenotypic and toms. Upon upper and lower endoscopy, the patient was molecular alterations has not been elucidated, and this found to have multiple polyps of the stomach, small bowel, disease is often overlooked or mistaken for other pol- and colon. The endoscopist noted that the polyps did not yposis syndromes. This case highlights the phenotypic look like “regular” adenomatous polyps. Several were overlap of MUTYH-associated polyposis with other resected from the stomach and small and large bowel. hereditary cancer syndromes, particularly attenuated The gastric polyps were ulcerated and contained dilated FAP, which complicates its recognition. In both condi- glands within inflamed stroma (Figure 6.14A–B). Some tions, patients typically present with 10 to 100 adeno- intestinal polyps were similar to the gastric polyps, con- mas (ie, attenuated polyposis), and some patients with taining enlarged, dilated glands with inspissated mucus MUTYH-associated polyposis have serrated polyps as and inflammation within prominent, markedly inflamed well. A significant minority of patients with MUTYH- stroma (Figure 6.14C–D). Other intestinal polyps con- associated polyposis also have, congenital hypertrophy tained irregular, crowded crypts with much less inflam- of retinal pigment epithelium, osteomas, and especially mation and intervening stroma (Figure 6.14E–F). Several duodenal adenomas, the latter of which are found in up of the gastric and the intestinal polyps contained foci of to 25%. Approximately 15% to 20% of affected patients dysplasia (Figure 6.14G–J). Upon questioning, the patient with MUTYH-associated polyposis have multiple ser- did not have a history of polyposis or cancer in his family, rated polyps and, in fact, meet diagnostic criteria for ser- but he was not a good historian. Genetic testing revealed rated polyposis. In addition, some patients present with a mutation in SMAD4, confirming juvenile polyposis colon cancer, typically at a relatively young age, in the syndrome. absence of overt polyposis; this presentation can mimic As previously discussed in the section on juvenile Lynch syndrome. MUTYH-associated polyposis should polyposis, the hamartomatous polyps of juvenile pol- be considered in patients with attenuated polyposis and yposis have an extensive differential diagnosis (particu- a family history suggestive of autosomal recessive inheri- larly the gastric polyps), including Peutz–Jeghers polyps, tance. In cases with apparent autosomal dominant inheri- inflammatory polyps, Cronkhite–Canada syndrome, tance, FAP should be considered first, though MUTYH and even Ménétrier disease. Genetic testing is impor- testing may be helpful in patients in whom an APC muta- tant not only for diagnostic purposes but also in deter- tion is not detected. mining future surveillance and risk assessment for both (A) (B) FIGURE 6.14 This gastric polyp from a patient with juvenile polyposis syndrome shows dilated, focally cystic glands containing inspissated mucus and inflammation, within inflamed stroma (A–B). (continued) 6 Approach to Hereditary Cancer Syndromes 137 (C) (D) (E) (F) (G) (H) FIGURE 6.14 (continued) The colonic polyps from the same patient show similar findings, including dilated glands and a prominent, markedly inflamed stroma (C–D). Other intestinal polyps contained irregular, crowded crypts with much less inflammation and intervening stroma (E–F). (continued) 13 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (I) (J) FIGURE 6.14 (continued) Several of the intestinal (G–H) and gastric (I–J) polyps contained foci of dysplasia. gastrointestinal and nongastrointestinal cancers in both challenges for pathologists, gastroenterologists, and the patient and their family members. It is also impor- surgeons. Indeed, our clinical colleagues are often sur- tant to note that only 20% to 50% of juvenile polyposis prised at the poor correlation between histologic findings syndrome patients will have a family history of polyps; and their endoscopic impressions and may have reserva- so lack of a history of polyposis does not exclude this tions regarding the value of prophylactic gastrectomy in diagnosis. affected patients. However, the lifetime risk for develop- ing gastric adenocarcinoma is extremely high (greater Case 3 than 80% by age 80), and women with CDH1 germline mutations also have an increased risk of developing lobu- A 35-year-old woman presented to her primary care lar breast cancer. Because of this increased risk, as well physician complaining of abdominal pain and nausea. as the difficulty in detecting early lesions in these patients She was initially treated with proton pump inhibitor (as illustrated by this case), prophylactic gastrectomy is therapy, but her symptoms did not improve, and in fact usually advised after age 20. Indications for genetic test- worsened. She was eventually referred to a gastroen- ing are somewhat controversial, but recent criteria sug- terologist. Upon endoscopy, the gastroenterologist did gest that the following scenarios should prompt genetic not see any definite lesions but took random biopsies testing for this disease: to rule out Helicobacter pylori. Biopsies showed small, subtle foci of in situ signet ring cell adenocarcinoma • Families with two or more cases of gastric cancer (Figure 6.15A–C). The patient’s maternal grandfather in first- or second-degree relatives, with one dif- had a history of gastric cancer, and she thought that one fuse cancer diagnosed in a patient younger than other maternal family member had a history of breast 50 years cancer. Mutational analysis revealed a germline muta- • Personal or family history of diffuse gastric cancer tion in the E-cadherin/CDH1 gene, and the patient was and lobular breast cancer, with one patient under the counseled regarding prophylactic gastrectomy. age of 50 Familial diffuse gastric carcinoma will be further • Three cases of diffuse gastric cancer in first- or sec- discussed in Chapter 8, but this case illustrates sev- ond-degree relatives, regardless of age eral important issues that are pertinent to the discus- • Patients diagnosed with diffuse gastric cancer before sion of hereditary cancer syndromes. This is a relatively age 40, especially in a low incidence population rare condition, that poses diagnostic and therapeutic (such as those in the United States and Canada) 6 Approach to Hereditary Cancer Syndromes 13 9 (A) (B) (C) FIGURE 6.15 Gastric biopsies in patients with CDH1 mutations often show multiple foci of in situ signet ring cell carcinoma, without corresponding macroscopically visible lesions (A–C). SELECTED REFERENCES Patel SG, Ahnen DJ. Familial colon cancer syndromes: an update of a rap- idly evolving field. Curr Gastroenterol Rep. 2012;14(5):428–438. Shia J, Yantiss RK. Molecular mechanisms of colorectal carcinogen- General esis. In: Yantiss RK, ed. Colorectal carcinoma and tumors of the Baron TH, Smyrk TC, Rex DK. Recommended intervals between screening vermiform appendix. Philadelphia, PA: Wolters Kluwer Lippincott and surveillance colonoscopies. Mayo Clin Proc. 2013;88(8):854–858. Williams & Wilkins; 2014:191–203. Burn J, Mathers J, Bishop DT. Genetics, inheritance and strategies for prevention in populations at high risk of colorectal cancer (CRC). Recent Results Cancer Res. 2013;191:157–183. Familial Adenomatous Polyposis and Hegde M, Ferber M, Mao R, Samowitz W, Ganguly A. ACMG technical Related Syndromes standards and guidelines for genetic testing for inherited colorec- tal cancer (Lynch syndrome, familial adenomatous polyposis, and Kerr SE, Thomas CB, Thibodeau SN, Ferber MJ, Halling KC. APC MYH-associated polyposis). Genet Med. 2014;16(1):101–116. germline mutations in individuals being evaluated for familial ade- Jasperson KW. Genetic testing by cancer site: colon (polyposis syn- nomatous polyposis: a review of the Mayo Clinic experience with dromes). Cancer J. 2012;18(4):328–333. 1591 consecutive tests. J Mol Diagn. 2013;15(1):31–43. Lucci-Cordisco E, Risio M, Venesio T, Genuardi M. The growing com- Liang J, Lin C, Hu F, et al. APC polymorphisms and the risk of colorec- plexity of the intestinal polyposis syndromes. Am J Med Genet A. tal neoplasia: a HuGE review and meta-analysis. Am J Epidemiol. 2013;161A(11):2777–2787. 2013;177(11):1169–1179. 14 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Pavicic W, Nieminen TT, Gylling A, et al. Promoter-specific alterations Hamartomatous Polyposes of APC are a rare cause for mutation-negative familial adenomatous polyposis. Genes, Chromosomes Cancer. 2014;53(10):857–864. Bennett KL, Mester J, Eng C. Germline epigenetic regulation of KILLIN in Cowden and Cowden-like syndrome. JAMA. MUTYH-Associated Polyposis 2010;304(24):2724–2731. Canzonieri C, Centenara L, Ornati F, et al. Endoscopic evaluation Aretz S, Uhlhaas S, Goergens H, et al. MUTYH-associated polyposis: of gastrointestinal tract in patients with hereditary hemorrhagic 70 of 71 patients with biallelic mutations present with an attenu- telangiectasia and correlation with their genotypes. Genet Med. ated or atypical phenotype. Intl J Cancer. 2006;119(4):807–814. 2014;16(1):3–10. Nielsen M, Franken PF, Reinards TH, et al. Multiplicity in polyp count Heald B, Mester J, Rybicki L, Orloff MS, Burke CA, Eng C. Frequent and extracolonic manifestations in 40 Dutch patients with MYH gastrointestinal polyps and colorectal adenocarcinomas in a associated polyposis coli (MAP). J Med Genet. 2005;42(9):e54. prospective series of PTEN mutation carriers. Gastroenterol. Nielsen M, Joerink-van de Beld MC, Jones N, et al. Analysis of MUTYH 2010;139(6):1927–1933. genotypes and colorectal phenotypes in patients With MUTYH- Hiljadnikova Bajro M, Sukarova-Angelovska E, Adelaide J, et al. A new associated polyposis. Gastroenterol. 2009;136(2):471–476. case with 10q23 interstitial deletion encompassing both PTEN and Vogt S, Jones N, Christian D, et al. Expanded extracolonic tumor spectrum BMPR1A narrows the genetic region deleted in juvenile polyposis in MUTYH-associated polyposis. Gastroenterol. 2009;137(6):1976– syndrome. J Appl Genet. 2013;54(1):43–47. 1985 e1–e10. Jee MJ, Yoon SM, Kim EJ, et al. A novel germline mutation in exon 10 of the SMAD4 gene in a familial juvenile polyposis. Gut Liver. Lynch Syndrome 2013;7(6):747–751. Lachlan KL. Cowden syndrome and the PTEN hamartoma tumor syn- Campbell PT, Curtin K, Ulrich CM, et al. Mismatch repair polymor- drome: how to define rare genetic syndromes. J Natl Cancer Inst. phisms and risk of colon cancer, tumour microsatellite instability 2013;105(21):1595–1597. and interactions with lifestyle factors. Gut. 2009;58(5):661–667. Latchford AR, Neale K, Phillips RK, Clark SK. Juvenile polyposis syn- Geiersbach KB, Samowitz WS. Microsatellite instability and colorectal drome: a study of genotype, phenotype, and long-term outcome. cancer. Arch Pathol Lab Med. 2011;135(10):1269–1277. Dis Col Rect. 2012;55(10):1038–1043. Gudgeon JM, Williams JL, Burt RW, et al. Lynch syndrome screening Marsh Durban V, Jansen M, Davies EJ, et al. Epithelial-specific loss of implementation: business analysis by a healthcare system. Am J PTEN results in colorectal juvenile polyp formation and invasive Managed Care. 2011;17(8):e288–e300. cancer. Am J Pathol. 2014;184(1):86–91. Jasperson KW, Samowitz WS, Burt RW. Constitutional mismatch Mester JL, Moore RA, Eng C. PTEN germline mutations in patients repair-deficiency syndrome presenting as colonic adenomatous initially tested for other hereditary cancer syndromes: would use polyposis: clues from the skin. Clin Genet. 2011;80(4):394–397. of risk assessment tools reduce genetic testing? The Oncologist. Kerber RA, Neklason DW, Samowitz

WS, Burt RW. Frequency of 2013;18(10):1083–1090. familial colon cancer and hereditary nonpolyposis colorectal can- Ni Y, Zbuk KM, Sadler T, et al. Germline mutations and variants in the cer (Lynch syndrome) in a large population database. Fam Cancer. succinate dehydrogenase genes in Cowden and Cowden-like syn- 2005;4(3):239–244. dromes. Am J Hum Genet. 2008;83(2):261–268. Rasmussen LJ, Heinen CD, Royer-Pokora B, et al. Pathological assess- Orloff MS, He X, Peterson C, et al. Germline PIK3CA and AKT1 muta- ment of mismatch repair gene variants in Lynch syndrome: past, tions in Cowden and Cowden-like syndromes. Am J Hum Genet. present, and future. Hum Mutat. 2012;33(12):1617–1625. 2013;92(1):76–80. Samowitz WS, Curtin K, Wolff RK, et al. Microsatellite instabil- Pilarski R, Burt R, Kohlman W, Pho L, Shannon KM, Swisher E. ity and survival in rectal cancer. Cancer Causes Control. Cowden syndrome and the PTEN hamartoma tumor syndrome: 2009;20(9):1763–1768. systematic review and revised diagnostic criteria. J Natl Cancer Shia J, Holck S, Depetris G, et al. Lynch syndrome-associated neo- Inst. 2013;105(21):1607–1616. plasms: a discussion on histopathology and immunohistochemis- Septer S, Zhang L, Lawson CE, et al. Aggressive juvenile polyposis in try. Fam Cancer. 2013;12(2):241–260. children with chromosome 10q23 deletion. World J Gastroenterol. Shia J, Stadler Z, Weiser MR, et al. Immunohistochemical staining for 2013;19(14):2286–2292. DNA mismatch repair proteins in intestinal tract carcinoma: how Tse JY, Wu S, Shinagare SA, et al. Peutz-Jeghers syndrome: a reliable are biopsy samples? Am J Surg Pathol. 2011;35(3):447–454. critical look at colonic Peutz-Jeghers polyps. Mod Pathol. Steinhagen E, Shia J, Markowitz AJ, et al. Systematic immunohisto- 2013;26(9):1235–1240. chemistry screening for Lynch syndrome in early age-of-onset Wain KE, Ellingson MS, McDonald J, et al. Appreciating the broad colorectal cancer patients undergoing surgical resection. J Am Coll clinical features of SMAD4 mutation carriers: a multicenter chart Surg. 2012;214(1):61–67. review. Genet Med. 2014;16(8):588–593. Steinke V, Holzapfel S, Loeffler M, et al. Evaluating the performance of White BD, Chien AJ, Dawson DW. Dysregulation of Wnt/beta- clinical criteria for predicting mismatch repair gene mutations in catenin signaling in gastrointestinal cancers. Gastroenterol. Lynch syndrome: a comprehensive analysis of 3,671 families. Int J 2012;142(2):219–232. Cancer. 2014;135(1):69–77. Yantiss RK. Hamartomatous polyps and polyposis syndromes. In: Vaughn CP, Baker CL, Samowitz WS, Swensen JJ. The frequency of Yantiss RK, ed. Colorectal carcinoma and tumors of the vermiform previously undetectable deletions involving 3′ exons of the PMS2 appendix. Philadelphia, PA: Wolters Kluwer Lippincott Williams & gene. Genes, Chromosomes Cancer. 2013;52(1):107–112. Wilkins; 2014:33–56. 7 Neoplasms of the Esophagus RHONDA K. YANTISS INTRODUCTION Glycogenic Acanthosis Glycogenic acanthosis appears as one or more white A variety of benign and malignant neoplasms occur in the mucosal plaques spanning only a few millimeters. Solitary esophagus; these can be broadly classified as epithelial, or scattered lesions are common and of no clinical impor- mesenchymal, and lymphoid proliferations (Table 7.1). tance, whereas diffuse esophageal glycogenic acanthosis In the esophagus, epithelial tumors (both benign and resembling candidiasis is a relatively specific manifesta- malignant) are far more common than those of mesen- tion of PTEN hamartoma tumor (Cowden) syndrome. chymal or lymphoid origin. Indeed, benign lymphoid Lesions reflect nodular expansion of the epithelium by proliferations and lymphomas are exceedingly rare pri- increased numbers of keratinocytes that contain abun- mary esophageal lesions; most cases represent secondary dant cytoplasmic glycogen, which can be highlighted esophageal involvement in the setting of systemic disease. with a periodic acid–Schiff (PAS) stain (Figure 7.1A–B). The purpose of this chapter is to discuss the most com- Glycogenic acanthosis is best appreciated when both mon tumors and tumor-like lesions of the esophagus. lesional and nonlesional tissues are included in the biopsy. Diseases that can affect any part of the gastrointestinal Occasionally, gastroenterologists may also mistake gly- tract, but infrequently affect the esophagus, are discussed cogenic acanthosis for the white plaques seen in eosino- elsewhere. philic esophagitis. INFLAMMATORY AND NON-NEOPLASTIC Squamous Papilloma LESIONS Squamous papillomas are very common and have a Gastroenterologists frequently identify and sample endo- reported prevalence of 1% in the general population, scopically apparent polyps and excrescences in the esoph- although it is likely that many reported cases are best agus, most of which are clinically inconsequential. Polyps, considered to be inflammatory-type polyps. Squamous plaques, and nodules of the proximal esophagus generally papillomas are classified as endophytic, exophytic, and reflect small islands of heterotopic tissue, whereas those spiked. Endophytic papillomas are almost exclusively of the distal esophagus tend to be reactive lesions related located in the distal esophagus. They are most common to gastroesophageal reflux disease or other forms of among patients with gastroesophageal reflux disease or esophagitis. The latter may contain squamous epithelium, other types of esophagitis, but are not consistently associ- glandular mucosa, or both, thereby mimicking either ated with human papillomavirus (HPV) infection. For this squamous or glandular neoplasms. For this reason, a brief reason, some pathologists classify these lesions as inflam- discussion of these entities is warranted. matory-type polyps, rather than papillomas, in order to 141 142 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 7.1 Classification of Esophageal Neoplasms of the basal zone, intercellular edema, and inflamed granu- According to the World Health Organization lation tissue in the lamina propria (Figure 7.2B). Exophytic papillomas contain broad, finger-like projec- Epithelial Tumors Mesenchymal Tumors tions of lamina propria lined by hyperplastic squamous epi- Premalignant lesions Granular cell tumor Squamous dysplasia (intraepithelial Hemangioma thelium. The cells display minimal, if any, cytologic atypia neoplasia) Leiomyoma and convincing viral cytopathic changes are typically lacking Low-grade Lipoma (Figure 7.2C). Historical studies demonstrated HPV DNA High-grade Gastrointestinal in nearly 80% of cases, although these older experiments Glandular dysplasia (intraepithelial stromal tumor neoplasia) Kaposi sarcoma may have been compromised by contaminant DNA in study Low-grade Leiomyosarcoma samples. More recent reports have failed to identify a clear High-grade Melanoma association between squamous papillomas and HPV. Similar Carcinoma Rhabdomyosarcoma to endophytic lesions, exophytic papillomas show a predi- Squamous cell carcinoma Synovial sarcoma Basaloid squamous cell carcinoma lection for the distal esophagus and are more commonly Spindle cell (sarcomatoid) carcinoma Lymphoma observed in patients with esophagitis. They may be multiple, Verrucous squamous cell carcinoma but do not have an established association with squamous Adenocarcinoma Secondary dysplasia or squamous cell carcinoma of the esophagus. Adenosquamous carcinoma malignancies Mucoepidermoid carcinoma Spiked papillomas are morphologically distinct and Adenoid cystic carcinoma comprise the least common subtype of squamous papil- Undifferentiated carcinoma loma. They may display hyperkeratosis with a verrucous appearance and occur anywhere in the esophagus (Figure (Neuro)Endocrine neoplasms Well-differentiated (neuro)endocrine 7.2D). Squamous hyperplasia with hyperkeratosis is typical tumor and some cases do show a prominent granular cell layer G1 with koilocytic features, similar to cutaneous verrucae. G2 Unlike other subtypes, spiked papillomas frequently harbor Poorly differentiated (neuro)endocrine carcinoma HPV DNA. They have not been associated with increased Large cell variant risk of squamous dysplasia or squamous cell carcinoma. Small cell variant Mixed adeno(neuro)endocrine carcinoma Infl ammatory-Type Polyp Most esophageal inflammatory-type polyps occur in avoid unnecessary concern on the part of the clinician and/ close proximity to the gastroesophageal junction. They or patient. Endophytic squamous papillomas have smooth, are frequently encountered in patients with esophagi- rounded contours and are surfaced by variably thick squa- tis, including erosive gastroesophageal reflux disease, mous epithelium that is usually inflamed or even ulcerated pill esophagitis, prior surgery, and infection. Those that (Figure 7.2A). The papillae are elongated with expansion develop in the squamous-lined esophagus have been (A) (B) FIGURE 7.1 Glycogenic acanthosis produces pale white mucosal plaques that reflect accumulation of glycogen within squamous epithelial cells. Enlarged cells have abundant colorless cytoplasm and expand the full thickness of the epithelium (A). PAS stain highlights the cytoplasmic glycogen (B). 7 Neoplasms of the Esophagus 14 3 (A) (B) (C) (D) FIGURE 7.2 Endophytic squamous papillomas have a nodular appearance and are composed of hyperplastic squamous mucosa with inflamed, edematous lamina propria (A). This endophytic papilloma shows mild reactive cytologic atypia and intraepithelial neutrophils (B). Exophytic squamous papillomas consist of frond-like projections of hyperplastic squamous epithelium and edematous lamina propria (C). Spiked squamous papillomas contain proliferating squamous cells with a verrucous appearance (D). termed “endophytic squamous papillomas,” as described to mid esophagus. Most of these lesions consist of gas- in the preceding paragraphs. Inflammatory-type polyps of tric mucosa. Clinicians refer to them as “inlet patches” the gastroesophageal junction and gastric cardia contain because they appear as pink plaques at the inlet of the glandular mucosa at the surface. These hyperplastic, or esophagus. Gastric heterotopias may contain antral-type regenerative, polyps contain an edematous lamina propria mucosa, oxyntic glands, or a combination of both. They covered by either hypermucinous or mucin-depleted fove- are generally asymptomatic, although large lesions may olar epithelium (Figure 7.3A–C). They may display a vil- secrete enough acid to produce heartburn, dysphagia, or lous or serrated appearance at low magnification, similar bleeding, and some become colonized by Helicobacter to hyperplastic/regenerative polyps of the gastric mucosa. pylori. Transformation to adenocarcinoma is extremely rare. Heterotopias may also consist of sebaceous glands (Figure 7.4); these lesions are known as Fordyce spots. Esophageal Heterotopias They appear as solitary yellow plaques or nodules owing At least 10% of patients undergoing upper endoscopic to the presence of parakeratotic debris overlying sebaceous evaluation have islands of ectopic mucosa in the proximal glands, as well as abundant lipid within glands. 14 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 7.3 Inflammatory polyps at the gastroesophageal junction resemble gastric hyperplastic polyps. Finger-like projections of inflamed lamina propria support hypermucinous epithelial cells (A–B). Slightly convoluted pits are enmeshed in inflamed lamina propria and lined by distended mucinous cells (C), and mucin-depleted foveolar epithelium may also be present on the polyp surface. Heterotopic pancreatic tissue is a common finding in its frequent occurrence among both pediatric patients and biopsy samples of the gastroesophageal junction, report- healthy individuals with normal pH probe studies argues edly present in up to 25% of normal patients. Pancreatic against this viewpoint. heterotopias consist of tightly packed lobules of acini lined by polarized cells with basally located nuclei and granular, luminally oriented cytoplasm (Figure 7.5A–B). EPITHELIAL NEOPLASMS Although this finding has historically been considered a metaplastic response to gastroesophageal reflux disease, Squamous Dysplasia and Squamous Cell Carcinoma Epidemiology and Pathogenesis Squamous cell carcinoma is the most common malignant tumor of the esophagus worldwide, yet its incidence var- ies depending on several factors. Most patients are older adults, and men are affected more frequently than women. Areas of highest incidence include China, Iran, South America, and South Africa, where risk is associated with lower socioeconomic status, dietary nitrates, and vitamin deficiencies. Risk factors for squamous cell carcinoma in Europe and the United States include alcohol use, smoking, and radiation, although the incidence of disease has been decreasing in these regions over the past several decades. Patients with squamous dysplasia, underlying achalasia, Plummer–Vinson syndrome, chronic esophagitis, or stric- tures are at particularly increased risk, as are those with tylosis palmaris et plantaris, a hereditary disorder result- FIGURE 7.4 This sebaceous heterotopia produced ing from a defect in RHBDF2, which encodes a serine an endoscopically evident yellow plaque in the protease important to epithelial integrity. Epidemiologic midesophagus. Lobules of mature sebaceous glands are features of squamous dysplasia are similar to those of present beneath the squamous mucosa. squamous cell carcinoma. 7 Neoplasms of the Esophagus 14 5 (A) (B) FIGURE 7.5 Pancreatic heterotopias are commonly encountered in biopsy samples of the gastroesophageal junction, but do not produce an endoscopically apparent lesion in most cases. Lobules of exocrine glands are associated with neutral mucin-containing glands (A). The cuboidal cells contain finely granular eosinophilic cytoplasm and, unlike parietal cells, are polarized with basally located nuclei (B). Early data suggested a causal role of HPV in the application, chromoendoscopy, narrow band imaging, and development of squamous cell carcinoma, although confocal laser endomicroscopy exploit properties unique this hypothesis has been largely disproven. More than to dysplasia and improve its endoscopic detection. 70 studies have evaluated the role of HPV in human esophageal carcinoma and many with positive results describe the presence of cutaneous viral types that are not oncogenic. None have demonstrated any r elationship between esophageal squamous cell carcinoma and other factors that may implicate HPV, such as sexual behav- ior, immunosuppression, and other HPV-related malig- nancies. Furthermore, recent studies performed under stringent conditions have failed to demonstrate a rela- tionship between HPV infection and either squamous cell carcinoma or

dysplasia, even in extremely high-risk areas of China. Thus, HPV likely plays a limited, if any, role in the development of esophageal squamous cell carcinoma. Clinical and Endoscopic Features Squamous dysplasia is asymptomatic; most cases are identified in patients who undergo endoscopic exami- nation for other reasons or undergo surveillance due to presumed cancer risk. Endoscopic manifestations of dys- plasia are often subtle and many cases are not visible by FIGURE 7.6 A 69-year-old male underwent surveillance white light examination. Some lesions appear as an ero- endoscopy for high-grade squamous dysplasia. Diffuse sion or area of erythema, whereas others cause slight mucosal abnormalities, including circumferential yellow– mucosal irregularity. Abnormal keratin or parakeratosis white plaques, extensively involve the midesophagus. on the surface of dysplasia can produce a white plaque or Biopsy sampling revealed low- and high-grade squamous patch (Figure 7.6). Enhancing techniques, such as iodine dysplasia. 14 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Invasive squamous cell carcinomas usually cause therapies are utilized in debilitated patients, as discussed obstructive-type symptoms, such as progressive dysphagia in subsequent sections. from solids to liquids, although occult bleeding and weight Tumor stage, as determined by the tumor, node, metas- loss are not uncommon. Slightly more than half (60%) tasis (TNM) staging system, is the most important prog- of cases occur in the midesophagus; those remaining are nostic factor among patients with esophageal squamous evenly distributed in the upper and lower esophagus. cell carcinoma. Tumors confined to the mucosa have a low Squamous carcinomas typically develop on a background (less than 5%) risk of regional lymph node metastasis, but of multifocal, or extensive, squamous dysplasia and pro- metastatic risk increases with progressive depth of inva- duce clinically evident mass lesions. Early cancers appear sion. Current data suggest that slightly more than 50% as nodules or plaques, whereas locally advanced tumors of tumors confined to the deep submucosa do not have circumferentially involve the esophageal wall and produce associated regional lymph node metastases and, thus, may luminal narrowing (Figure 7.7A–B). Some specific sub- be treated with endoscopic resection in selected patients. types of squamous cell carcinoma characteristically pro- These early lesions are usually detected in regions of high- duce polypoid luminal masses, as discussed in the section disease prevalence where screening and surveillance pro- on macroscopic and microscopic features of squamous cell grams are widely utilized. Unfortunately, such programs carcinoma. in the United States are limited to select patient groups due to the low prevalence of the disease. As a result, most Management and Prognostic Factors patients in this country have locally advanced disease at the time of diagnosis and may not be surgical candidates. Squamous dysplasia, especially high-grade dysplasia, is a Overall 5-year survival rates for esophageal squamous cell major risk factor for the development of invasive squa- carcinoma range from 5% to 10% in the United States and mous cell carcinoma. Patients with low-grade dysplasia improve to only 35% among patients treated with cura- have a cancer risk 3- to 8-fold higher than that of the gen- tive surgical resection. In a subset of patients, resectability eral population, and the risk increases to 28- to 34-fold and overall survival may be improved through the use of among patients with high-grade dysplasia. Thus, foci of neoadjuvant therapy. dysplasia are removed or ablated and followed by endo- scopic surveillance. Improved endoscopic techniques, such as endoscopic mucosal resection, are increasingly Macroscopic and Microscopic Features used to remove relatively large lesions and, as a result, Squamous dysplasia is generally treated by endoscopic esophagectomy is generally reserved for patients who techniques, rather than surgery, so most resection speci- are not candidates for endoscopic management. Ablative mens consist of small pieces of mucosa and a variable (A) (B) FIGURE 7.7 An early squamous cell carcinoma appears as an umbilicated nodule (A, arrow). The background mucosa shows multifocal dysplasia characterized by numerous white plaques. A more advanced lesion is nearly circumferential and narrows the esophageal lumen. The tumor is surfaced by gray–white plaques and displays stigmata of recent hemorrhage (B). 7 Neoplasms of the Esophagus 147 amount of submucosa. These specimens are often oriented system. Low-grade dysplasia is defined by the presence of by gastroenterologists to ensure optimal tissue processing neoplastic cells limited to the lower half of the epithelial and histologic evaluation. The deep and lateral aspects of thickness, whereas the presence of neoplastic cells occupy- endoscopic excisional specimens represent resection mar- ing more than 50% of the epithelial thickness is classified gins that should be inked prior to perpendicular section- as high-grade dysplasia (Figure 7.9B–C). The latter cate- ing of the tissue. Dysplastic foci are also frequently present gory encompasses squamous cell carcinoma in situ, which in esophagectomy specimens that contain invasive squa- is no longer a preferred term in gastrointestinal neoplasia. mous cell carcinoma. Similar to their endoscopic appear- Epidermoid metaplasia has been recently described as a ances, dysplastic areas may be erythematous, ulcerated, harbinger of squamous cell carcinoma in the esophagus, and or white, reflecting abnormal accumulation of keratin likened to leukoplakia of the oral mucosa. Lesions typically (Figure 7.8). Some cases have a diffuse corrugated appear- develop in patients with risk factors for esophageal squa- ance that reflects abnormal keratinization and expansion mous cell carcinoma, namely exposure to tobacco smoke of the mucosal thickness. and alcohol, and nearly 20% of patients have concomitant At low power, there is an abrupt transition between squamous dysplasia or carcinoma at endoscopy. Epidermoid foci of dysplasia and adjacent non-neoplastic mucosa in metaplasia may also be observed in resected esophagi con- histologic sections (Figure 7.9A). Features of dysplasia taining invasive squamous cell carcinoma. Lesions are include crowded, immature cells with nuclear enlarge- sharply demarcated from adjacent normal mucosa and char- ment, nuclear membrane irregularities, hyperchromasia, acterized by squamous hyperplasia, expansion of the basal and increased mitotic activity. Most cases do not show zone, acanthosis, prominence of the granular cell layer, and striking keratinization, but some have overlying ortho- or hyperorthokeratosis (Figure 7.9D). Although its prognostic parakeratosis and show intense cytoplasmic eosinophilia. significance is not clear at this point, epidermoid metapla- Squamous dysplasia is graded based on a two-tiered sia may represent a mature-appearing variant of dysplasia, similar to that occurring in the oropharynx. The gross appearance of squamous cell carcinoma varies depending on the stage of the disease. Early tumors appear as small, pearly white nodules or ulcers (Figure 7.10A–B). Superficial cancers may be removed by endo- scopic mucosal resection or endoscopic submucosal dissection. Similar to endoscopically removed foci of squamous dysplasia, the deep and lateral aspects of exci- sion specimens should be denoted as resection margins at the time of gross examination, and serially sectioned per- pendicularly to the deep margin. Endoscopically removed carcinomas are submitted entirely for histologic evalua- tion and the depth of invasion into the submucosa and proximity of the tumor to the margin reported. Multifocal and advanced tumors may be treated with esophagec- tomy (Figure 7.10C), provided the disease is limited to the esophagus and regional lymph nodes. Resected cancers are characteristically fungating, nearly circumferential masses with surface ulceration and nodularity, although those treated with neoadjuvant therapy may show some regression in the form of mural fibrosis or a residual ulcer (Figure 7.10D). Tumors should be sampled to assess the depth of invasion as well as the extent of therapeutic response in cases treated with neoadjuvant therapy. The microscopic features of esophageal squamous cell carcinoma are similar to those of squamous carcinomas at other sites. Irregular sheets and nests of overtly malig- nant cells are associated with keratin pearls and desmo- FIGURE 7.8 This archival resection specimen contains plastic stroma (Figure 7.11A). Tumor cells are typically extensive low- and high-grade squamous dysplasia. The large and contain abundant, densely eosinophilic cyto- area of high-grade dysplasia is erythematous (arrow). The plasm. Nuclei are centrally located and display irregu- background mucosa is slightly thick, reflecting squamous lar contours, coarse chromatin, and prominent nucleoli. hyperplasia. Mitotic figures, including abnormal ones, are readily 14 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 7.9 Squamous dysplasia (right) is sharply demarcated from the adjacent nondysplastic squamous epithelium (A, arrows). Lesional cells contain enlarged, hyperchromatic nuclei. In low-grade dysplasia, the dysplastic cells are limited to the lower half of the squamous mucosa. The basal layer is disorganized, and increased mitotic activity is present up to the midlevel of the epithelium (B). High-grade dysplasia displays full-thickness cytologic atypia with numerous mitotic figures (C). Epidermoid metaplasia consists of mature-appearing epithelium with an abnormal granular cell layer and parakeratosis (D). apparent and cellular necrosis is frequently present. Not There are several morphologic variants of esopha- uncommonly, basaloid cells with hyperchromatic nuclei geal squamous cell carcinoma, but all have similar clini- and scant cytoplasm occupy the peripheries of tumor cell cal features, treatment, and stage-dependent outcomes. nests and seem to mature toward their centers. High- Extremely well-differentiated verrucous carcinomas fea- grade features include the presence of single infiltrating ture minimal nuclear atypia and well-developed acantho- cells, delicate trabeculae of neoplastic cells, and sheet-like sis with hyperkeratosis (Figure 7.12A–C). These tumors growth of nonkeratinizing, basaloid cells (Figure 7.11B). can be difficult to recognize because invasion occurs Striking pleomorphism and multinucleated cells may be across a broad smooth front, rather than overtly infiltrat- encountered. Of note, tumors treated with neoadjuvant ing single cells and clusters, and thus may not be appar- therapy should be assessed for therapeutic response, as ent in superficial biopsy samples. High-grade variants greater tumor regression is associated with better survival. include basaloid squamous cell carcinoma and spindle However, nonviable tumor (ie, keratin debris and fibrosis) cell (sarcomatoid) carcinoma. The former show a pre- in the esophagus or regional lymph nodes should not be dilection for the mid- and distal esophagus and contain counted toward tumor extent when assigning TNM stage, tumor cells with large nuclei, finely granular chromatin, as discussed subsequently. small nucleoli, and scant cytoplasm arranged in lobules, 7 Neoplasms of the Esophagus 14 9 (A) (B) (C) (D) FIGURE 7.10 An early invasive squamous cell carcinoma forms an irregular, pearly-white nodule in the squamous mucosa above the level of the gastroesophageal junction (A). Another example of squamous cell carcinoma forms an irregular plaque (arrow) on a background of squamous dysplasia (B). An advanced cancer forms an ulcerated mass (arrow) in the upper esophagus (C). Neoadjuvant treatment may cause tumor regression, leaving behind an ulcer on a background of atrophic mucosa (D). nests, and cords (Figure 7.12D–E). Lobules frequently closely resemble fibroblasts in some cases. They contain display peripheral nuclear palisading and central necro- ovoid, or elongated, nuclei and bipolar cytoplasmic tails. sis that simulate the appearance of high-grade neuro- Nucleoli and nuclear hyperchromasia are variable, and endocrine carcinoma. They may also show a cribriform pleomorphic tumor giant cells are seen in some cases. growth pattern and contain faintly eosinophilic, hyalin- Spindle cell carcinomas may also contain bone, cartilage, ized, basement membrane-like material reminiscent of or other heterologous elements that raise the possibility of adenoid cystic carcinoma (Figure 7.12E). In fact, most a sarcoma. However, primary osteosarcoma, chondrosar- reported cases of esophageal adenoid cystic carcinoma coma, and rhabdomyosarcoma are exceedingly rare in the likely represent basaloid squamous cell carcinomas. esophagus, so consideration for spindle cell carcinoma Spindle cell carcinoma (Figure 7.12F–I) is an unusual must be given prior to rendering a diagnosis of sarcoma. variant of squamous cell carcinoma that typically grows as a polypoid, intraluminal mass, rather than an infiltra- Ancillary Studies tive lesion. Variable numbers of epithelioid and spindle cells comprise the tumors, although epithelioid cells may Squamous dysplasia and squamous cell carcinoma show be scarce or limited to the in situ component. Spindle cells similar molecular abnormalities, consistent with the theory display a spectrum of cytologic abnormalities and may that squamous carcinogenesis is a progressive, multistep 15 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 7.11 A well-differentiated squamous cell carcinoma displays keratin pearls in the invasive component (A). Note the associated desmoplastic stroma. A higher grade squamous cell carcinoma infiltrates as single cells, cords, and small nests (B). (A) (B) (C) (D) FIGURE 7.12 (continued) 7 Neoplasms of the Esophagus 151 (E) (F) (G) (H) (I) FIGURE 7.12 (continued) Extremely well-differentiated verrucous carcinomas pose diagnostic challenges in biopsy samples. This patient had a nearly circumferential, fungating mass, but biopsy samples showed only an extremely

well-differentiated proliferation of squamous cells with overlying thick parakeratosis (A). Thick, irregular projections of squamous cells extend from the deep aspect of the epithelium and scattered dyskeratotic cells are present (arrow) in an abnormal location (B). Enlarged, but mature, squamous cells are also present in this region, some of which show mitotic activity (arrow) and nuclear enlargement (C). Basaloid squamous cell carcinomas are high-grade malignancies that often show sheet-like growth (D). However, some cases contain round aggregates of tumor cells associated with basement membrane-like material that simulates adenoid cystic carcinoma (E). Esophageal spindle cell carcinomas are composed of a diffuse proliferation of malignant spindle cells that may resemble a sarcoma (F–G). Plump, stellate tumor cells with enlarged, hyperchromatic nuclei undermine the mucosa (H). Similar to spindle cell carcinomas at other sites, these tumors may show an absence, or near lack, of keratin staining (I). process. Early changes present in dysplasia include altera- cell carcinoma, but none have proven to be clinically u seful tions in telomerase activity, loss of heterozygosity (LOH), for diagnostic or therapeutic purposes. increased cyclin D1 expression, and hypermethylation of INK4/CDKNA (p16), whereas TP53 mutations accom- pany onset of high-grade cytologic abnormalities. Diagnostic Challenges Increased expression of epidermal growth factor receptor Biopsy samples from patients with inflammatory condi- (EGFR) has been described in at least 50% of carcinomas tions of the esophagus may contain epithelial regenerative and abnormal expression of cell cycle regulatory proteins changes that mimic dysplasia, particularly when ulcers are is common. Studies have implicated other biomarkers as present. Squamous cell hyperplasia, nuclear enlargement important players in the evolution of esophageal squamous and hyperchromasia, nucleolar prominence, and increased 15 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 7.13 Reactive squamous epithelial changes may mimic low-grade dysplasia. Cytologic atypia tends to be more pronounced near the base of the epithelium in reactive mucosa (A). Mitotic figures are increased, but basal keratinocytes maintain their polarity and show maturation (B). mitotic activity are all features of reparative atypia that A more problematic issue is the failure to recognize mimic dysplasia. These inflammatory changes tend to be invasive squamous cell carcinoma in superficial biopsy uniform and show a gradual transition to more normal- samples. Well-differentiated carcinomas may show a appearing areas, whereas dysplasia is sharply demarcated papillary growth pattern with apparent surface matura- from adjacent non-neoplastic mucosa. Benign epithelial tion that simulates the appearance of a squamous papil- hyperplasia shows regular papillary elongation and pro- loma. Such tumors often have a broad, pushing type of trusions of basal keratinocytes into the lamina propria, invasion that does not elicit a striking desmoplastic stro- and reactive atypia is largely confined to the basal epithe- mal response. They also lack substantial mitotic activ- lium (Figure 7.13A–B). Most reparative changes that are ity beyond the basal regions of the epithelium, and show severe enough to mimic dysplasia are also associated with minimal cytologic atypia (Figure 7.16A–B). Fortunately, intraepithelial neutrophilic inflammation. In contrast, the interface between dysplastic squamous epithelium and the lamina propria tends to be irregular with broad, or fused, projections from the base of the epithelium. Cytologic atypia is heterogeneous, varying from one cell to the next, and intraepithelial inflammation is less common than in reactive atypia. Chemotherapeutic agents and radiation can produce severe abnormalities that simulate neoplasia, including nuclear enlargement and hyperchromasia with membrane irregularities. However, the epithelium usually appears attenuated, rather than proliferative, and affected cells contain vacuolated cytoplasm while maintaining their nuclear-to-cytoplasmic ratios (Figure 7.14). The diagnosis of invasive squamous cell carcinoma is generally straightforward, although challenges occur in some situations. “Pseudoepitheliomatous” hyperplasia adjacent to erosions or overlying a granular cell tumor is a well-recognized mimic of squamous cell carcinoma, espe- cially in superficial biopsies (Figure 7.15A–B). Features FIGURE 7.14 Chemotherapy induces cytologic atypia favoring a diagnosis of benign hyperplasia include the that may simulate neoplasia. However, the epithelium is presence of only mild cytologic atypia and confinement of attenuated, rather than proliferative, and atypical epithelial squamous cell nests to the superficial mucosa. cells contain abundant cytoplasm. 7 Neoplasms of the Esophagus 15 3 (A) (B) FIGURE 7.15 Granular cell tumors of the esophagus, as in other sites, are associated with pseudoepitheliomatous hyperplasia. Irregular nests of keratinizing squamous epithelium are present in the lamina propria (A). The subjacent granular cell tumor contains cells with abundant, faintly eosinophilic granular cytoplasm and pyknotic nuclei. Tumor cells are intimately associated with dense collagen bands (B). the clinical appearance of such lesions is not at all that Finally, biopsy samples from an ulcer may contain of a squamous papilloma, at least in most cases. Well- unusually large endothelial cells and fibroblasts with an differentiated carcinomas tend to be quite large, destruc- epithelioid or stellate appearance. These activated stromal tive plaque-like lesions that may be circumferential, cells contain abundant cytoplasm, as well as large nuclei whereas papillomas appear as small verrucous plaques and prominent nucleoli, and lack the clustered appearance that rarely exceed 1.0 cm in diameter. Carcinomas also of carcinoma cells (Figure 7.17A). Biopsy samples of ulcers display an irregular interface with the subjacent stroma may also contain sheets of activated lymphocytes, mac- and show abnormal keratinization in the deep epithelium. rophages, and necrotic granulocytes in adherent exudates (A) (B) FIGURE 7.16 This biopsy from a patient with dysphagia was initially interpreted as an atypical squamous papilloma before the macroscopic description of a large, destructive mass lesion was obtained. At low power, the tumor cells show surface maturation, and lack an infiltrating growth pattern (A). At higher power, there is focal loss of nuclear polarity, but the surface maturation is maintained and the broad, pushing invasive front is deceptively bland (B). 15 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 7.17 Biopsy samples of an esophageal ulcer contain activated fibroblasts and endothelial cells with enlarged, slightly hyperchromatic nuclei with smooth contours dispersed in granulation tissue (A). Ulcer debris contains necrotic inflammatory cells that may simulate the appearance of lymphoma. This case also contains embedded crystalline iron reflecting a drug-related injury (B). that can be misinterpreted as lymphoma. However, the epithelium in the distal esophagus, even if intestinal meta- atypical cells do not infiltrate the tissue and often con- plasia is lacking (ie, columnar-lined esophagus). The over- tain small, convoluted nuclei, even in degenerated material all cancer risk of patients with intestinal metaplasia in the (Figure 7.17B). esophagus is less than 2% and increases with the extent of the disease. However, evidence substantiating the biologic Adenocarcinoma, Dysplasia, and Barrett risk of nongoblet glandular metaplasia is not yet available. Esophagus For this reason, most practitioners in the United States still require goblet cells to establish a diagnosis of Barrett Barrett Esophagus esophagus. Patients with Barrett esophagus are usually Barrett esophagus is relatively common, affecting nearly treated with aggressive antireflux medications followed 6% in the general population and 11% of patients with by regular surveillance endoscopy with four-quadrant a history of heartburn. Males are affected more than sampling every 1 to 2 cm of the length of affected mucosa. females and prevalence increases with age. Risk fac- Barrett esophagus is classified as long (greater than tors include gastroesophageal reflux disease, hiatal her- 3 cm), short (1–3 cm), and ultrashort (less than 1 cm) seg- nia, central obesity, delayed esophageal clearance, and ments, depending upon its extent. Small areas of meta- decreased resting tone of the lower esophageal sphincter. plasia near the gastroesophageal junction may appear Barrett esophagus is a premalignant condition character- as a mucosal irregularity or produce no abnormalities, ized by replacement of the normal squamous epithelium making it difficult to establish a diagnosis of ultrashort by non-neoplastic, but genetically unstable, glandular segment Barrett esophagus. Extensive metaplasia pro- mucosa in the distal esophagus. Persistent gastroesopha- duces salmon-pink, velvety mucosal tongues that ema- geal reflux disease leads to inflammation-related injury nate from the gastroesophageal junction into the distal and cumulative molecular events in the metaplastic epi- esophagus (Figure 7.18A). Corresponding biopsy sam- thelium, placing the patient at risk for progressive dyspla- ples contain goblet cells interspersed on a background sia and adenocarcinoma. The American Gastroenterology of nongoblet columnar (ie, foveolar) epithelial cells filled Association defines Barrett esophagus as (a) the presence with faintly eosinophilic neutral mucin (Figure 7.18B). of endoscopically apparent metaplasia of the distal esoph- Occasional oxyntic glands, absorptive cells, Paneth cells, agus (columnar-lined esophagus) that (b) shows intesti- and endocrine cells may be present, and the mucosa is nal metaplasia (goblet cells) on histology. However, this often inflamed. Importantly, many patients with Barrett definition is not accepted in some countries, including the esophagus develop duplication of the muscularis mucosae, United Kingdom and Japan, where a diagnosis of Barrett in which case the most superficial layer consists of haphaz- esophagus can be based on the presence of any glandular ardly arranged discontinuous bundles of smooth muscle 7 Neoplasms of the Esophagus 15 5 (A) (B) FIGURE 7.18 Barrett esophagus appears as erythematous tongues of glandular mucosa that emanate into the distal esophagus (A). Gray–blue, barrel-shaped goblet cells are interspersed in a background of foveolar-type epithelium (B). cells and lies within lamina propria. The deeper, second Epidemiology and Pathogenesis of layer is better organized with compact bundles of smooth Barrett-Esophagus-Related Neoplasia muscle cells (Figure 7.19A–B). This layer represents the true muscularis mucosae and delineates the deepest aspect There has been a six-fold increase in the incidence of esoph- of the mucosa. Failure to recognize duplication of the mus- ageal adenocarcinoma over the past three decades, as well cularis mucosae may pose a problem to pathologists when as a seven-fold increase in disease-related mortality during assessing for depth of invasion in biopsies of early adeno- the same time period. Virtually all cases of glandular dys- carcinomas, as the thickened muscularis mucosae may be plasia and invasive adenocarcinoma develop in patients mistaken for muscularis propria. with Barrett esophagus, and thus the epidemiology of (A) (B) FIGURE 7.19 Barrett esophagus often displays duplication of the muscularis mucosae. In this example, submucosal glands are present deep to an organized layer of smooth muscle cells, which represents the true muscularis mucosae. A thick band of smooth muscle cells is also present directly subjacent to the mucosa (A). Higher magnification of the same area demonstrates the haphazard arrangement of smooth muscle cells in the duplicated muscularis mucosae. This layer is present in the lamina propria (B). 15 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide these disorders is the same as that of Barrett esophagus. as chromoendoscopy with indigo carmine, high-resolution Rare tumors derived from gastric heterotopias and sub- magnification with narrow band imaging, and confocal mucosal glands do occur, but these lesions are so rare laser endomicroscopy, enhance detection of dysplasia with that their representation in the literature consists of case improved sensitivity and specificity. Some cases of glandu- reports and extremely small series. Carcinomas associated lar dysplasia produce subtle areas of erythema or raised with Barrett esophagus typically occur in older adults, plaques that can also be better visualized with enhancing and males are affected more than females. The presence techniques. Early cancers usually produce an endoscopi- of glandular dysplasia is the most important risk factor for cally evident abnormality, such as a polypoid or plaque- cancer development in patients with Barrett esophagus. like configuration, ulcer, or a subtle mucosal irregularity (Figure 7.21A–B). The presence of an ulcer, polyp, or plaque Clinical and Endoscopic Features of Barrett in the context of Barrett esophagus is much more likely to Esophagus and Glandular Neoplasia represent a cancer than dysplasia. Advanced tumors form bulky, friable masses that show ulceration or obstruct the Although patients with Barrett esophagus may have symp- lumen (Figure 7.21B). toms related to chronic gastroesophageal reflux disease, metaplasia and dysplasia of the esophageal mucosa are asymptomatic. As a result, many patients with Barrett Management and Prognostic Features esophagus or dysplasia in the United States never partici- Cancer risk increases with progressive dysplasia among pate in surveillance programs. Such individuals only come patients with Barrett esophagus: approximately 5% to to clinical attention when they develop symptoms related to 10% of patients with low-grade dysplasia develop carci- advanced adenocarcinoma, such as progressive dysphagia to noma, compared to 15% to 30% of patients with high- solids and liquids, pain, and weight loss. Indeed, approxi- grade dysplasia. Treatment of dysplasia generally consists mately 80% of patients in this country

have locally advanced of endoscopic mucosal resection, although a variety of cancers and 50% have metastases at the time of diagnosis. ablative techniques may be considered in debilitated or Glandular dysplasia occurs in a multifocal fashion, elderly patients. These include photodynamic therapy, reflecting a field effect in Barrett mucosa. Most foci of radiofrequency ablation, laser ablation, and argon plasma dysplasia are endoscopically inapparent by white light coagulation. Esophagectomy is limited to specific situa- examination, and because of this surveillance protocols tions as determined by patient circumstances. have historically called for random four-quadrant muco- Slightly more than 20% of submucosally invasive sal biopsies every 2 cm from the gastroesophageal junc- tumors metastasize to regional lymph nodes, compared to tion to the neo-squamocolumnar junction (Figure 7.20). only 1.3% of tumors confined to the mucosa. For this rea- Recent advances in endoscopic imaging techniques, such son, early lesions limited to the superficial submucosa can be managed with endoscopic mucosal resection, whereas cancers that infiltrate the deeper submucosa may be ame- nable to endoscopic submucosal dissection. Survival of patients with invasive adenocarcinoma is determined by tumor stage. Five-year survival rates exceed 80% among tumors limited to the mucosa or submucosa without regional lymph node metastases, but patients with unre- sectable disease have a poor prognosis with 5-year sur- vival rates of less than 5%. Treatment of esophageal adenocarcinoma is stage-dependent. Virtually all patients with locally advanced disease receive preoperative neoad- juvant therapy to improve resectability and survival. Macroscopic and Microscopic Features of Barrett- Esophagus-Related Neoplasia Glandular Dysplasia Dysplasia is defined as a neoplastic proliferation of epithe- FIGURE 7.20 This patient with long segment Barrett lial cells confined to the basement membrane of the epithe- esophagus underwent thermal ablation of the metaplastic lium in which it developed. Biopsy samples from patients mucosa. At the time of the procedure, he was noted to with Barrett esophagus are designated as negative for dys- have a small ulcer (arrow), which was sampled and proved plasia, positive for dysplasia, or indefinite for dysplasia. to be high-grade dysplasia. Cases classified as positive for dysplasia are graded using 7 Neoplasms of the Esophagus 157 (A) (B) FIGURE 7.21 This elderly patient was found to have an irregular nodule (arrow) on a background of Barrett esophagus (asterisk). Biopsy and subsequent endoscopic submucosal dissection revealed a superficially invasive cancer (A). Another patient with dysphagia was found to have a nearly obstructing friable mass in the distal esophagus that proved to be a high-grade invasive adenocarcinoma (B). a two-tiered system (low- and high-grade dysplasia). Cases dysplasia are characterized by the presence of the same with cytologic atypia that do not reach the threshold of dys- population of atypical cells in both deep glands and sur- plasia, or for which limited sampling or a background of face epithelium, and a concomitant lack of surface matu- inflammation are confounding factors, may be considered ration. Most cases of dysplasia display an overall increase indefinite for dysplasia. Most problematic cases labeled as in gland density at low magnification that reflects their indefinite for dysplasia show cytologic atypia limited to the proliferative nature, although the crowding is generally crypts, or atypical features in mucosa that is also inflamed. mild in cases of low-grade dysplasia. Most cases of low- There is often a sharp demarcation between foci of grade dysplasia have an intestinal phenotype with cyto- dysplasia and adjacent nondysplastic epithelium. Foci of logic abnormalities similar to those seen in intestinal (A) (B) FIGURE 7.22 Foci of low-grade dysplasia are typically sharply demarcated from the background glands of Barrett esophagus (A). Crowded, mucin-depleted cells are present at the surface and show mild cytologic atypia that is morphologically similar to intestinal adenomas. Enlarged, hyperchromatic nuclei maintain their polarity with respect to the basement membrane (B). A small amount of active inflammation is present, but is not enough to account for the cytologic abnormalities. 15 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide adenomas (Figure 7.22A). Dysplastic cells contain ovoid open or dispersed chromatin, and conspicuous nucleoli or elongated hyperchromatic nuclei with membrane irreg- (Figure 7.23C–D). Mitotic figures, including atypical ularities, but maintain their polarity in low-grade dyspla- ones, and necrotic cellular debris are common and may sia (Figure 7.22B). be seen at all levels within the mucosa (Figure 7.23E). The High-grade dysplasia shows severe cytologic atypia distinction between high-grade dysplasia and intramu- often in combination with architectural abnormalities of cosal carcinoma in the setting of Barrett esophagus can the dysplastic glands. Most cases have an intestinal phe- be very challenging, and is discussed in the subsequent notype and consist of crowded, irregularly shaped glands, Diagnostic Challenges section. cribriform glands, and/or villous or papillary projec- The term “foveolar dysplasia” refers to cases with a tions on the surface (Figure 7.23A–B). Cytologically, the papillary or “tufted” growth pattern at the surface and/ cells of high-grade dysplasia have a complete loss of cell or dilated glands, or microglands, in the deep mucosa polarity, pronounced nuclear membrane irregularities, (Figure 7.24). The dysplastic cells in these cases are (A) (B) (C) (D) (E) FIGURE 7.23 High-grade dysplasia typically features both architecturally abnormal glands and high-grade cytologic atypia (A). Architectural abnormalities include irregularly branched, fused, and dilated glands, as well as microlumina or cribriform glands (B). Closer examination of this area demonstrates high-grade nuclear abnormalities, including round nuclei with dispersed chromatin, irregular membranes, and prominent nucleoli. Many cells have lost their polarity to the basement membrane (C–D). Some glands may contain necrotic luminal debris (E). 7 Neoplasms of the Esophagus 15 9 setting of inflammatory bowel disease than a sporadic adenoma. Neoplastic polyps that develop in Barrett esophagus are better classified as polypoid dysplasias. The term “adenoma” should be reserved for the (very rare) benign neoplasm derived from submucosal glands in the esophagus or lesions that develop near the gastro- esophageal junction in the setting of familial adenoma- tous polyposis. Glandular dysplasia is increasingly treated by endo- scopic techniques, and thus resection specimens generally consist of excisional specimens rather than esophagectomy specimens. The deep and lateral aspects of these specimens comprise the resection margins, and they should be inked prior to processing, similar to excisional specimens for squamous dysplasia, as previously described in that sec- tion. Specimens should be submitted entirely for histologic FIGURE 7.24 Cases of foveolar dysplasia feature a evaluation. Esophagectomy is rarely performed for high- highly atypical proliferation of cells at the mucosal surface. grade dysplasia alone in the current era. The nuclei are large and ovoid with irregular contours, open chromatin, and macronucleoli, and the cytoplasm is often eosinophilic. Mitotic figures are readily identified. Esophageal Adenocarcinoma Currently, the majority of superficially invasive adenocar- cinomas are managed endoscopically, so esophagectomies polygonal, with eosinophilic or clear cytoplasm. They are usually performed for advanced tumors (Figure 7.26A). typically contain large round nuclei with irregular con- Locally advanced adenocarcinomas may be treated with tours, vesicular chromatin, and macronucleoli, and are neoadjuvant chemoradiotherapy in an effort to improve often designated as high-grade dysplasia. tumor resectability and overall survival. Similar to rectal “Crypt dysplasia” is a term used to define cyto- carcinomas, neoadjuvant treatment can cause substantial logic atypia (often high-grade) confined to the deep tumor regression that may produce an apparent ulcer sur- glands, in association with apparent surface maturation rounded by dense fibrosis (Figure 7.26B). We recommend (Figure 7.25A–D). This finding has been postulated to submitting the entire ulcerated area with perpendicular represent a precursor to invasive adenocarcinoma, simi- sections to the radial (adventitial) margin to evaluate for lar to other types of dysplasia. However, most available residual tumor. However, cases that show an essentially data regarding the biologic importance of this finding are unaltered tumor mass or an obvious carcinoma are evalu- based on small retrospective studies that include a dispro- ated with representative sections of the tumor and margin. portionate number of high-risk patients with concomitant Most invasive esophageal adenocarcinomas have an overt dysplasia or carcinoma. To date, there are no con- intestinal phenotype, or at least demonstrate prominent trolled prospective studies assessing the significance of glandular differentiation (Figure 7.27A–D). Variants crypt dysplasia in patients with Barrett esophagus. From a including signet ring cell carcinoma, mucinous carcinoma, practical standpoint, problematic cases with marked cyto- and lymphoepithelioma-like carcinoma (see the section on logic atypia limited to the glands may be designated as uncommon types of esophageal adenocarcinoma) can also indefinite for dysplasia. However, most pathologists con- develop at this site, although they are much less common. sider low-grade atypia confined to the crypts to be nega- Most of these less common variants display at least some tive for dysplasia. It is important to remember that true degree of sheet-like growth, and are usually considered high-grade dysplasia may occasionally be confined to the high-grade neoplasms. deep glands, and lack overlying surface maturation. Treated tumor cells often show striking degenerative Barrett adenoma is a historical term used to denote cytologic atypia that should not be taken into account when the presence of polypoid dysplasia in the setting of assigning tumor grade. Regressed tumors may also contain Barrett esophagus. Such lesions often have an intestinal acellular mucin pools in the esophageal wall or regional phenotype similar to tubular or villous adenomas of the lymph nodes (Figure 7.28), although only viable tumor cells colorectum, and may contain areas of low- or high-grade should be considered when assigning pathologic tumor dysplasia. This terminology has fallen out of favor in stage. Documentation of tumor regression is important in the modern era because such lesions arise from a back- these cases because it is predictive of survival; extensive ground of “at-risk” Barrett mucosa or even multifocal regression is associated with improved survival, whereas a dysplasia, which is more analogous to dysplasia in the limited tumor response predicts a poorer outcome. 16 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 7.25 These biopsies of Barrett esophagus contain atypical glands in the deep mucosa that are associated with mature epithelium at the luminal surface (A–B). High magnification demonstrates marked cytologic atypia in the deep glands. The nuclei are large and hyperchromatic, but maintain their polarity to the basement membrane (C–D). Many pathologists would designate findings such as these as indefinite for dysplasia, although these changes have also been termed “crypt dysplasia.” Ancillary Studies p16 through INK4/CDKNA methylation are associated with progression to high-grade dysplasia and/or esopha- As previously mentioned, Barrett esophagus contains geal adenocarcinoma, but none of these biomarkers are genetically unstable epithelial cells. Lesional epithelium superior to histologic evaluation in predicting progression harbors a variety of molecular abnormalities despite its of Barrett esophagus. Some investigators have shown that morphologic similarities to gastric and intestinal epithe- immunohistochemical stains for AMACR and p53 may lium. The majority (90%) of cases show INK4/CDKNA identify cases of low-grade dysplasia that are more likely (p16) alterations. Aneuploid cell populations, cytogenetic to progress to high-grade dysplasia or cancer, but these abnormalities, LOH, DNA methylation, and TP53 muta- markers are of limited value in distinguishing nondysplas- tions may also be detected, even in cases that lack dyspla- tic glandular epithelium from dysplasia. sia. Aneuploidy, LOH at 17p and 9p, and inactivation of 7 Neoplasms of the Esophagus 161 (A) (B) FIGURE 7.26 This esophagectomy specimen contains a large, friable mass located above the gastroesophageal junction. A small amount of residual Barrett mucosa is present at the right (A, arrow). Neoadjuvant therapy induces tumor regression. Resection specimens usually lack a luminal mass, but show ulcers in association with mural fibrosis (B). (A) (B) (C) (D) FIGURE 7.27 Invasive adenocarcinomas of the esophagus usually show glandular differentiation. This resection specimen shows a poorly differentiated adenocarcinoma, in which some degree of gland formation is still visible, beneath an area of extensive high-grade dysplasia (A). Note the thickened muscularis mucosae. A second example features irregular, infiltrating glands extending from the mucosa into the muscularis propria (B). At high power, this example contains both glands and single infiltrating tumor cells (C). This high-grade carcinoma contains numerous infiltrating signet ring cells, in addition to glands (D). 16 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide overexpress HER2. This receptor is targeted by trastu- zumab, a humanized monoclonal antibody that binds its extracellular domain. Treatment with this agent in combi- nation with conventional chemotherapy results in a 26% reduced risk of cancer-related death compared to conven- tional therapy alone. Other emerging targeted therapies include

the EGFR inhibitor cetuximab and lapatinib, an orally administered small molecule tyrosine kinase inhibi- tor with activity against HER2 and EGFR. Bevacizumab, sunitinib, and sorafenib are new agents with anti-VEGF activity that may have applications in the future. Diagnostic Challenges Biopsy samples from Barrett esophagus often display repar- ative cytologic changes due to persistent gastroesophageal FIGURE 7.28 Neoadjuvant therapy causes a variable reflux disease which, in some cases, may mimic dysplasia. degree of tumor regression. Residual pools of acellular Features of regenerative atypia include variable mucin mucin are present in the esophageal wall of this case. depletion, slight nuclear enlargement with dense chroma- tin, prominent nucleoli, and increased mitotic activity. In contrast to dysplasia, however, regenerative changes are Many esophageal adenocarcinomas harbor molecu- usually more pronounced in the deep glands compared to lar alterations that represent potential targets of directed the surface epithelium, and show a gradual (rather than medical therapies that have a synergistic effect when sharply demarcated) transition to regions that are clearly used in combination with conventional adjuvant therapy. non-neoplastic. In general, one may consider a diagno- These tumors frequently overexpress transmembrane sis of “indefinite for dysplasia” when cytologic atypia is tyrosine kinase growth factor receptors, including those limited to the gland region (see also preceding section on of the EGFR family, such as HER2. Approximately 25% crypt dysplasia), or when abundant neutrophilic inflam- to 30% of esophageal and gastroesophageal junctional mation is present in immature-appearing surface epithe- adenocarcinomas, especially intestinal-type tumors, lium (Figure 7.29A–B). However, complex architectural (A) (B) FIGURE 7.29 This case of Barrett esophagus shows a mildly atypical proliferation of glandular epithelium at the luminal surface with associated neutrophilic inflammation (A). Another case displays cytologically atypical cells in the deep glands that meet criteria of low-grade dysplasia, but are associated with mature epithelium on the surface (B). Both cases may be considered to be indefinite for dysplasia. 7 Neoplasms of the Esophagus 16 3 abnormalities cannot be adequately explained by either a plastic cap during the procedure. Cautery on the deep and location within the deep gland region or the presence of lateral margins may cause challenges when evaluating for neutrophils. Thus, fused or cribriform glands, papillary or adequacy of excision as well. Assessing the depth of tumor micropapillary projections, and microglands are evidence invasion in these limited specimens can also be particu- of high-grade dysplasia, regardless of inflammation or con- larly problematic when the muscularis mucosae is dupli- finement of atypical epithelium to the deep mucosa. cated. Penetration through the deeper layer is required Recognition of invasive carcinoma within the mucosa in order to render a diagnosis of submucosal invasion, is important because these tumors have a small but defi- whereas tumors that transgress the duplicated superficial nite risk of spread through lymphatic vessels to regional layer are classified as intramucosal cancers. lymph nodes. For this reason, intramucosal carcinoma of the esophagus is assigned a pathologic stage of pT1a, Less Common Types of Esophageal Carcinoma whereas similar lesions in the colon are regarded as essen- tially equivalent to carcinoma in situ. Some esophageal carcinomas resemble those of salivary The distinction between high-grade glandular dys- gland origin, particularly adenoid cystic or mucoepider- plasia and invasive adenocarcinoma can be challenging, moid carcinomas, and have been labeled as such in the especially in biopsy samples. Superficial biopsies of inva- older literature. Although it is possible that salivary-type sive adenocarcinoma generally do not show desmoplasia, tumors could arise from submucosal glands, most cancers because tumors rarely elicit a stromal reaction when they with these features develop in association with Barrett invade the mucosa. However, there are several features of esophagus or squamous neoplasia and contain elements intramucosal carcinoma in the esophagus that serve as of adenocarcinoma or squamous cell carcinoma. The vast helpful diagnostic clues (Figure 7.30A–F). Architecturally majority of esophageal tumors classified as adenoid cys- complex anastomosing or cribriform glands that expand tic carcinoma are more appropriately considered to be the lamina propria are highly suggestive of carcinoma, as high-grade, basaloid squamous carcinomas and pursue a are single infiltrating tumor cells in the lamina propria clinical course comparable to that of squamous cell carci- and angulated glands that lack a connection to clearly noma; biologically indolent adenoid cystic carcinomas are noninvasive glands. Pagetoid spread of tumor cells in the exceedingly rare in the esophagus. Similarly, cancers that adjacent, non-neoplastic squamous mucosa can be a very display divergent differentiation with glandular and squa- helpful distinguishing feature as well when the differential mous elements do not behave in an indolent fashion typical diagnosis includes high-grade dysplasia and adenocarci- of mucoepidermoid carcinoma. Most of these lesions are noma, as this finding is not seen in high-grade dysplasia. probably best considered as adenosquamous carcinomas Recently, two groups have published criteria advocat- because they pursue a clinical course comparable to that of ing for a category intermediate between high-grade dyspla- esophageal adenocarcinoma and squamous cell carcinoma sia and intramucosal carcinoma, referred to as “high-grade (Figure 7.31). Lymphoepithelioma-like carcinomas of the dysplasia with marked glandular architectural distortion, esophagus contain syncytial sheets of malignant cells inti- cannot exclude intramucosal carcinoma” or “high-grade mately admixed with mononuclear cell-rich infiltrates and dysplasia with features ‘suspicious’ for invasive carci- tumor infiltrating lymphocytes. The neoplastic cells har- noma.” These authors suggest that cases with three or bor large nuclei with thick, irregular membranes and one more glands with luminal debris or single cell infiltration or more prominent nucleoli, as well as faintly eosinophilic of the lamina propria are more likely to have invasive ade- cytoplasm. Some lymphoepithelioma-like carcinomas con- nocarcinoma on resection. However, this designation has tain areas of either squamous cell carcinoma or adenocar- not been widely accepted in general practice. cinoma and may show microsatellite instability. Pagetoid The differential diagnosis of invasive carcinoma in the spread of tumor cells in non-neoplastic squamous epithe- submucosa is more straightforward. Dysplasia may colo- lium of the esophagus can occur in patients with invasive nize deep mucosal and submucosal esophageal glands and squamous cell carcinoma, adenocarcinoma, or Paget dis- simulate the appearance of invasive adenocarcinoma in ease derived from submucosal gland ducts. rare cases. Unlike invasive adenocarcinoma in the submu- cosa, colonization of glands by dysplasia does not show a destructive growth pattern or a desmoplastic tissue reac- Neuroendocrine Tumors of the Esophagus tion, and the benign submucosal glands are tightly clus- Early investigators postulated that the diffuse endocrine tered in lobules. system of the gastrointestinal tract was derived from the The increased use of advanced endoscopic techniques neural crest and, thus, classified tumors composed of simi- has led to greater expectations of pathologists with lar cells as neuroendocrine neoplasms. Others later noted respect to tumor reporting in small specimens. Excisional that these cells, and tumors that simulated their appear- specimens often show artifactual erosion of the surface ance, contained ultrastructural dense core granules, which epithelium that results from suction of the sample into a they interpreted to reflect a “neurosecretory” nature, and 16 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 7.30 Features that suggest the presence of intramucosal carcinoma include complex, fused, and irregularly shaped glands, sometimes referred to as the “never-ending gland pattern,” (A–C), detached, proliferating glands with dilation and luminal necrotic debris (D), single infiltrating tumor cells (E), and pagetoid spread of cells in squamous epithelium (F). showed a “neuroendocrine” phenotype with immunoposi- normal secretory function of these hormone- producing tivity for chromogranin A, neuron specific enolase, CD56, cells. Importantly, chromogranin A is a prohormone that is and synaptophysin. We now know that endocrine cells of cleaved to produce functionally active peptides, so it is bet- the gastrointestinal tract have no relationship to the neu- ter considered a marker of endocrine, rather than neural, ral crest. They are derived from epithelial stem cells with differentiation. It is interesting that, while many authors potential for divergent differentiation into endocrine cells, accept that endocrine cells comprise the diffuse endocrine Paneth cells, goblet cells, and absorptive cells, and neo- system of the gastrointestinal tract, they advocate “neuro- plasms with similar features are also derived from mul- endocrine” to denote neoplasms that are phenotypically tipotent stem cells. Endocrine cells of the gastrointestinal similar. At this point, “endocrine” and “neuroendocrine” tract contain dense core secretory granules that reflect the are used synonymously to describe gastrointestinal tumors 7 Neoplasms of the Esophagus 16 5 cellular necrosis is readily identified. Immunostains for Ki-67 demonstrate a large proportion of positive tumor cells, often approaching 80%. Some cases are associated with a variable component of invasive squamous cell car- cinoma or high-grade squamous dysplasia in the surface epithelium. The major differential diagnosis in these types of tumors is extension into the esophagus from a small cell carcinoma of the lung, although occasionally the dys- cohesive nature of these tumors raises the possibility of lymphoma. GRADING AND STAGING OF ESOPHAGEAL CARCINOMA The World Health Organization and TNM staging system FIGURE 7.31 Adenosquamous carcinomas contain nests of squamoid tumor cells admixed with areas of do not provide useful guidance to pathologists regarding glandular differentiation. grade assignment to esophageal carcinomas. Some sub- types, including lymphoepithelioma-like, basaloid squa- mous, spindle cell (sarcomatoid), signet ring cell, mucinous, and neuroendocrine carcinomas, are generally regarded as that elaborate hormones and express endocrine markers high-grade malignancies, but criteria for grading of con- and synaptophysin. ventional squamous cell carcinoma and adenocarcinoma Primary neuroendocrine tumors of the esophagus are are lacking. This issue is particularly problematic with uncommon, and account for less than 1% of epithelial respect to pathologic stage classification since tumor grade tumors at this site. Similar to other organs of the gastroin- is incorporated into the overall stage assignment for both testinal tract, they are classified as well-differentiated and tumor subtypes. In our practice, we generally limit the poorly differentiated based upon the extent to which the definition of well-differentiated squamous cell carcinoma tumor cells resemble endocrine cells normally present in the to examples of verrucous carcinoma and those that are gastrointestinal tract (see also Chapter 3). By convention, mostly keratinizing with few basaloid cells. With respect well-differentiated tumors are subclassified as low (G1) or to adenocarcinomas, we endorse a two-tiered system intermediate (G2) grade based on a combination of mitotic that combines well-to-moderately differentiated (G1–G2) activity (less than 2 versus 2–10 mitotic figures/10 high- tumors in the low-grade category and groups those with power fields) and Ki-67 immunolabeling (less than 3% less than 25% glandular differentiation as high-grade (G3– versus 3%–20% immunopositivity). However, well-differ- G4), similar to grading systems applied to other gastroin- entiated neuroendocrine tumors of the esophagus are so testinal malignancies. uncommon that their clinicopathologic features have not Pathologic tumor staging of esophageal carcinoma is been well described. Like similar tumors of the stomach performed in accordance with the TNM staging manual and intestines, they display an organoid, or nested, growth of the American Joint Committee on Cancer (Table 7.2). pattern of tumor cells within collagenous stroma. Tumor Similar to the rest of the gastrointestinal tract, the local cells contain pale, or faintly eosinophilic, cytoplasm and extent of the disease is assessed in the T category, whereas round nuclei with stippled chromatin and small nucleoli. regional lymph node and distant metastases are classified Poorly differentiated neuroendocrine tumors are more in the N and M categories, respectively. Although this stag- common than well-differentiated tumors in the esophagus. ing system assigns a tumor stage to dysplasia (pTis), many These bulky, exophytic malignancies are high-grade (G3) pathologists do not include this information in pathol- carcinomas that typically present at an advanced stage in ogy reports of endoscopic mucosal resections, as the bio- older adults (particularly males) and pursue an aggressive logic risk of this finding is essentially nil provided that the course with a mean survival of less than 1 year. Most of lesional area is completely removed. these tumors have a small cell phenotype similar to their Although the same criteria are used for pathologic counterparts in the lung, and contain nests and sheets of stage assessment of squamous cell carcinoma and ade- overtly malignant cells with scant cytoplasm and angu- nocarcinoma, the overall stage groupings of these enti- lated, irregular nuclei, although large cell variants also ties are separate. Stage groupings for invasive squamous occur (Figure 7.32A–C). Mitotic activity is generally brisk cell carcinoma incorporate information regarding the (greater than 20

mitotic figures/10 high-power fields) and primary tumor stage, regional lymph nodes, and distant 16 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 7.32 This high-grade neuroendocrine carcinoma of the esophagus has a sheet-like growth pattern and extensive necrosis. The overlying surface is completely ulcerated (A). At higher power, the tumor contains sheets of dyscohesive tumor cells with large round nuclei, stippled chromatin, and numerous mitotic figures, and a rim of amphophilic cytoplasm (B). Necrosis and karyorrhectic nuclear debris are common, and numerous mitoses are easily seen (C). TABLE 7.2 Pathologic Staging Criteria of Esophageal and Gastroesophageal Junctional Carcinomas Primary tumor (pT) TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis High-grade dysplasia T1a Tumor invades lamina propria or muscularis mucosae T1b Tumor invades submucosa T2 Tumor invades muscularis propria T3 Tumor invades adventitia T4 Tumor invades adjacent structures T4a Resectable tumor invading pleura, pericardium, or diaphragm T4b Unresectable tumor invading other adjacent structures Regional lymph nodes (pN) NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastases N1 Metastasis in 1–2 regional lymph nodes N2 Metastasis in 3–6 regional lymph nodes N3 Metastasis in 7 or more regional lymph nodes Distant metastases (pM) M0 No distant metastases M1 Distant metastases 7 Neoplasms of the Esophagus 167 TABLE 7.3 Anatomic Stage Groupings of Esophageal Squamous Cell Carcinoma Stage T N M Grade Location 0 Tis N0 M0 1, X Any IA T1 N0 M0 1, X Any IB T1 N0 M0 2, 3 Any T2, T3 N0 M0 1, X Lower, X IIA T2, T3 N0 M0 1, X Upper, Middle T2, T3 N0 M0 2, 3 Lower, X IIB T2, T3 N0 M0 2, 3 Upper, Middle T1, T2 N1 M0 Any Any IIIA T1, T2 N2 M0 Any Any T3 N1 M0 Any Any T4a N0 M0 Any Any IIIB T3 N2 M0 Any Any IIIC T4a N1, N2 M0 Any Any T4b Any M0 Any Any Any N3 M0 Any Any IV Any Any M1 Any Any metastases as well as anatomic tumor location and tumor Tumor invasion of the mucosa (pT1a) can be subclassified grade (Table 7.3). Well-differentiated squamous cell carci- based on tumor extent: m1 denotes neoplasia confined to noma is generally assigned a lower overall stage compared the basement membrane (ie, high-grade dysplasia), m2 is to moderate-to-poorly differentiated tumors in cases with- defined as tumor limited to the lamina propria, and m3 out metastatic disease. Similarly, overall stage groupings of describes tumor that invades, but is limited to, the mus- esophageal adenocarcinoma also incorporate tumor grade cularis mucosae. Although it lacks anatomic landmarks, (Table 7.4). The distinction between low-grade (G1–G2) invasion of the submucosa (pT1b) can be similarly divided and high-grade (G3–G4) is considered most relevant, so into inner (sm1), middle (sm2), and outer (sm3) thirds. the use of either a two- or four-tiered system is appropriate. The increased use of endoscopic techniques for the management of dysplasia and early invasive carcinoma MESENCHYMAL TUMORS OF has resulted in a shift among pathologists with respect to THE ESOPHAGUS pathologic reporting of endoscopically removed lesions. As it turns out, the depth of tumor invasion in the mucosa Granular Cell Tumor and submucosa predicts the likelihood of regional lymph node metastasis of both squamous cell carcinoma and Granular cell tumors may occur anywhere in the gastroin- adenocarcinoma. Thus, pathologists should report the testinal tract, but are most common in the esophagus (see depth of tumor invasion in endoscopic excision specimens. also Chapter 5). Benign tumors are often multifocal, and TABLE 7.4 Anatomic Stage Groupings of Esophageal Adenocarcinoma Stage T N M Grade 0 Tis N0 M0 1, X IA T1 N0 M0 1, 2, X IB T1 N0 M0 3 T2 N0 M0 1, 2, X IIA T2 N0 M0 3 IIB T3 N0 M0 Any T1, T2 N1 M0 Any IIIA T1, T2 N2 M0 Any T3 N1 M0 Any T4a N0 M0 Any IIIB T3 N2 M0 Any IIIC T4a N1, N2 M0 Any T4b Any M0 Any Any N3 M0 Any IV Any Any M1 Any 16 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide can be deeply infiltrative, but these features do not imply Historic data suggested that nearly 80% occurred in the malignancy. Granular cell tumors usually occur in older distal esophagus and arose from the inner circular layer adults, are slightly more common in women, and affect of the muscularis propria, whereas only 20% developed African Americans more frequently than Caucasians. from the muscularis mucosae. However, more recent expe- Most tumors are incidentally discovered at the time of rience in the endoscopic era suggests that tumors of the endoscopy and appear as firm, pale yellow polyps or muscularis mucosae are more common than previously plaques limited to the mucosa and submucosa of the distal believed. Tiny multifocal tumors have been termed seed- esophagus. However, large lesions that extensively involve ling leiomyomata, and multiple large tumors are consid- the muscularis propria may cause dysphagia. ered to represent leiomyomatosis. Tumors derived from the Granular cell tumors consist of sheets and nests of cells muscularis propria produce irregular thickening and may with infiltrative borders that are intimately associated with attain a large size that causes symptoms of obstruction. thick bands of collagen (Figure 7.15B). Tumor cells are Leiomyomas have smooth, rounded contours and homo- closely related to Schwann cells and show strong, diffuse geneous echogenicity by endoscopic ultrasound. Small S100 immunopositivity. They contain abundant faintly tumors of the muscularis mucosae can be endoscopically eosinophilic, granular cytoplasm that reflects the pres- excised, whereas larger lesions are treated with surgical ence of myelin-filled lysosomes. Occasional PASD-positive enucleation. Esophagectomy is reserved for patients with eosinophilic globules are present in most cases. Granular extremely large, or multiple, tumors. cell tumors display minimal cytologic atypia; nuclei may Leiomyomas are firm, unencapsulated nodules with a appear to be pyknotic and mitotic figures are sparse. Some pale, whorled cut surface. Paucicellular fascicles of bland granular cell tumors elicit a peculiar pattern of pseudo- smooth muscle cells intersect at right angles. The tumor epitheliomatous squamous hyperplasia in the overlying cells contain densely eosinophilic cytoplasm and ovoid, epithelium, which may simulate the appearance of inva- blunt-ended nuclei (Figure 7.33A–B). A small amount of sive squamous cell carcinoma. For this reason, one should coagulative necrosis may be present in large tumors, but carefully examine the lamina propria and submucosa any substantial necrosis or mitotic activity should raise before rendering a diagnosis of squamous cell carcinoma, concern for a leiomyosarcoma. Tumor cells show strong especially in cases without a clinical suspicion for malig- diffuse immunohistochemical staining for actins, desmin, nancy. Malignant granular cell tumors are extremely rare and caldesmon, but are uniformly negative for KIT. Some and show atypical cytologic features, nuclear enlargement, tumors do contain KIT-positive mast cells that should not increased mitotic activity, and cellular necrosis, similar to be misinterpreted as evidence of a gastrointestinal stromal malignant mesenchymal neoplasms elsewhere. tumor (Figure 7.33B). Gastrointestinal Stromal Tumor Leiomyosarcoma Less than 1% of all gastrointestinal stromal tumors arise Many reports of esophageal leiomyosarcoma predate the from the esophagus. These tumors usually occur in older recognition of gastrointestinal stromal tumor, which is adults and cause symptoms of obstruction, dysphagia, or now believed to account for most malignant mesenchy- occult blood loss. Most esophageal stromal tumors are bio- mal tumors of this site. Leiomyosarcoma is a tumor that logically aggressive with high-risk features including large develops almost exclusively in older adults. Most cases are size and relatively brisk mitotic activity (greater than 5 high-grade spindle cell neoplasms with overt features of mitoses/50 high-power fields). They have a fleshy appear- malignancy, including brisk mitotic activity, cellular necro- ance with hemorrhage, necrosis, and cystic degeneration, sis, and severe cytologic atypia. Esophageal tumors share a rather than the firm, whorled appearance of a leiomyoma. similar immunophenotype with leiomyomas and are nega- Gastrointestinal stromal tumors are highly cellular with tive for KIT. long, sweeping fascicles of spindle cells that intersect at oblique angles. Lesional cells contain abundant eosino- Giant Fibrovascular Polyp philic cytoplasm and tapered nuclei with coarse chromatin and show strong immunopositivity for KIT and DOG1. Fibrovascular polyps are intriguing, but rare, lesions The topic of gastrointestinal stromal tumors is discussed of the proximal esophagus. Virtually all cases occur in further in Chapter 5. adults and originate near the cricopharyngeus muscle. Presumably, polyp formation is the result of prolapsing redundant mucosal folds that become increasingly dis- Leiomyoma and Leiomyomatosis torted due to elevated intraluminal pressures in the upper Leiomyomas are the most common mesenchymal tumors esophageal sphincter region. Most patients complain of of the esophagus. They occur equally among men and progressive dysphagia that reflects gradual enlargement women and are multifocal in nearly 25% of patients. of the polyp. However, some polyps are clinically silent 7 Neoplasms of the Esophagus 16 9 (A) (B) FIGURE 7.33 Leiomyomas contain sweeping fascicles of bland smooth muscle cells and display negligible mitotic activity (A). They do contain mast cells that show immunopositivity for KIT (B), which should not be mistaken as evidence of a GIST. until they obstruct the larynx, or are regurgitated into the intervening stroma. Cuboidal epithelial cells harbor neu- posterior hypopharynx. Such cases produce symptoms of tral mucin and basally oriented nuclei with minimal cyto- acute respiratory distress, vomiting, belching, coughing, logic atypia. Unlike hyperplastic polyps, pyloric gland or even sudden death by asphyxiation. adenomas show strong immunohistochemical staining for Fibrovascular polyps have a sausage-like, multilobu- MUC6 and contain foveolar epithelial cells that are largely lated appearance. They tend to be large and protrude into limited to the polyp surface. Preliminary data suggest that the lumen on a broad stalk. The squamous mucosal surface at least 50% of pyloric gland adenomas near the gastro- is often inflamed or eroded, owing to local trauma, and the esophageal junction develop in association with intestinal lamina propria and submucosa contain variable amounts metaplasia, many of which can show dysplasia. of loose, edematous myxoid tissue, extracellular collagen, and mature adipose tissue (Figure 7.34A–D). Most polyps are inflamed and harbor proliferating fibroblasts, some of Metastases to the Esophagus which may contain bizarre nuclei with prominent nucle- Secondary malignancies involve the esophagus by direct oli, especially in areas of ulceration. Vessels are promi- extension from adjacent organs or hematogenous metas- nent, and often have thick walls and surrounding fibrosis. tasis. Most commonly, carcinomas of the respiratory Fibrovascular polyps are benign and cured by simple exci- tract invade the thoracic esophagus, causing symptoms of sion. Of note, some atypical lipomas may develop in the dysphagia, hematemesis, or pneumonitis due to fistuliz- esophagus and closely simulate the appearance of a giant ing disease. These tumors typically display squamous or fibrovascular polyp. glandular differentiation. Invasive squamous cell carcino- mas of pulmonary origin are morphologically and immu- nohistochemically indistinguishable from those of the OTHER ESOPHAGEAL NEOPLASMS esophagus, although the differential diagnosis between pulmonary and esophageal adenocarcinoma can usually Pyloric Gland Adenoma be resolved. Tumor location above the level of the gastro- Pyloric gland adenomas rarely develop at the gastroesoph- esophageal junction and an absence of Barrett esophagus ageal junction where they can form esophageal polyps, are useful pathologic features that should suggest second- but they are more commonly encountered in the stomach. ary involvement of the esophagus by adenocarcinoma. Most are incidentally discovered in patients undergoing Immunostains for pulmonary markers, such as TTF-1, are endoscopic evaluation for symptoms related to the upper typically negative in esophageal adenocarcinomas. Napsin gastrointestinal tract. Pyloric gland adenomas contain does not reliably distinguish between tumors of pulmo- tightly packed lobules of small round glands with little nary and esophageal origin. 170 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 7.34 Giant fibrovascular polyps contain both mesenchymal and epithelial elements. This polyp contains abundant fat and fibrous tissue with chronic inflammation, lymphoid aggregates, and normal squamous epithelium (A–B; A, courtesy of Dr. Elizabeth Montgomery). Vessels are prominent, and may be thick-walled with surrounding fibrosis (C). Occasional atypical stromal stellate cells are seen, enmeshed in collagen (D, courtesy of Dr. Elizabeth Montgomery). Any advanced malignancy may metastasize to the Malignant Melanoma esophagus, although esophageal metastases are much less Primary malignant melanoma of the esophagus is common than those to other organs of the tubular gut. extremely rare and accounts for less than 0.1% of

all Patients with metastatic disease typically present with esophageal neoplasms. Patients are older adults who symptoms similar to those of individuals with primary present with progressive dysphagia, obstruction, hemor- malignancies, namely dysphagia, coughing, vomiting, and rhage, cough, or pain due to locally advanced tumors. occult blood loss or hematemesis. Tumors that show a pre- The prognosis is dismal with a mean survival of less than dilection for the gastrointestinal tract include carcinomas 1 year. Esophageal melanoma is unassociated with any of the lung, breast, and kidney, as well as malignant mela- identifiable risk factors and has no apparent relationship noma. Features suggesting the possibility of a metastasis with cutaneous melanoma. The background mucosa include tumor multifocality, location of adenocarcinoma may show melanosis, although a causal role for this find- in the upper or midesophagus, and the presence of exten- ing has not been established. Tumors typically appear sive lymphovascular invasion in biopsy samples. 7 Neoplasms of the Esophagus 171 Abraham SC, Singh VK, Yardley JH, Wu TT. Hyperplastic pol- yps of the esophagus and esophagogastric junction: histologic and clinicopathologic findings. Am J Surg Pathol. 2001;25(9): 1180–1187. Akiyama J, Bertele A, Brock C, et al. Benign and precursor lesions in the esophagus. Ann NY Acad Sci. 2014;1325(1):226–241. Squamous Dysplasia and Squamous Cell Carcinoma Cao W, Chen X, Dai H, et al. Mutational spectra of p53 in geographi- cally localized esophageal squamous cell carcinoma groups in China. Cancer. 2004 15;101(4):834–844. Denlinger CE, Thompson RK. Molecular basis of esophageal cancer development and progression. Surg Clin North Am. 2012;92(5):1089–1103. Devlin S, Falck V, Urbanski SJ, et al. Verrucous carcinoma of the esophagus eluding multiple sets of endoscopic biopsies and endo- scopic ultrasound: a case report and review of the literature. Can J FIGURE 7.35 This primary malignant melanoma of the Gastroenterol. 2004;18(7):459–462. Kwatra KS, Prabhakar BR, Jain S, Grewal JS. Sarcomatoid carcinoma esophagus contains sheets of neoplastic cells, some of (carcinosarcoma) of the esophagus with extensive areas of osse- which are pigmented. Lesional cells contain abundant ous differentiation: a case report. Indian J Pathol Microbiol. cytoplasm and have a vaguely plasmacytoid appearance, 2003;46(1):49–51. although they show nuclear variability. Lauwers GY, Grant LD, Scott GV, et al. Spindle cell squamous carcinoma of the esophagus: analysis of ploidy and tumor prolif- erative activity in a series of 13 cases. Hum Pathol. 1998;29(8): 863–868. as a polypoid intraluminal mass. Similar to malignant Liu H, Li YQ, Yu T, et al. Confocal endomicroscopy for in vivo detec- melanoma of other organs, esophageal tumors may be tion of microvascular architecture in normal and malignant lesions pigmented or amelanotic, and display either spindle or of upper gastrointestinal tract. J Gastroenterol and Hepatol. epithelioid cells with an organoid arrangement. Tumor 2008;23(1):56–61. Metzger R, Schneider PM, Warnecke-Eberz U, et al. Molecular biology cells contain eosinophilic or amphophilic cytoplasm of esophageal cancer. Onkologie. 2004;27(2):200–206. and large, often eccentric nuclei that impart a plasma- Singhi AD, Arnold CA, Crowder CD, et al. Esophageal leukoplakia or cytoid appearance. Intranuclear inclusions and nucleoli epidermoid metaplasia: a clinicopathological study of 18 patients. may be prominent (Figure 7.35). Esophageal melanomas Mod Pathol. 2014;27(1):38–43. Teng H, Li X, Liu X, et al. The absence of human papillomavirus in express melanocytic markers, including S100, A103, esophageal squamous cell carcinoma in East China. Int J Clin Exp and HMB-45, although high-grade and spindle cell Pathol. 2014;7(7):4184–4193. lesions may show loss of immunostaining for one, or Uedo N, Fujishiro M, Goda K, et al. Role of narrow band imaging for more, antibodies. diagnosis of early-stage esophagogastric cancer: current consensus of experienced endoscopists in Asia-Pacific region. Dig Endosc. 2011;23 Suppl 1:58–71. Wang GQ, Abnet CC, Shen Q, et al. Histological precursors of oesoph- SELECTED REFERENCES ageal squamous cell carcinoma: results from a 13 year prospective follow up study in a high risk population. Gut. 2005;54(2):187–192. Zhang XH, Sun GQ, Zhou XJ, et al. Basaloid squamous carcinoma of General esophagus:a clinicopathological, immunohistochemical and elec- Barker N, Clevers H. Lineage tracing in the intestinal epithelium. Curr tron microscopic study of sixteen cases. World J Gastroenterol. Protoc Stem Cell Biol. 2010;Chapter 5:Unit5A 4. 1998;4(5):397–403. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. Rice TW, Blackstone EH, Rusch VW. Esophagus and esophagogastric Adenocarcinoma, Glandular Dysplasia, and junction. In: Edge SB, Byrd DR, Compton CC, Fritz AG, Greene Barrett Esophagus FL, Trotti A, eds. AJCC Cancer Staging Manual. 7th ed. New Abraham SC, Krasinskas AM, Correa AM, et al. Duplication of the York, NY: Springer;2010:103–115. muscularis mucosae in Barrett esophagus: an underrecognized fea- Wang KK, Prasad G, Tian J. Endoscopic mucosal resection and endo- ture and its implication for staging of adenocarcinoma. Am J Surg scopic submucosal dissection in esophageal and gastric cancers. Pathol. 2007;31(11):1719–1725. Curr Opin Gastroenterol. 2010;26(5):453–458. Anaparthy R, Sharma P. Progression of Barrett oesophagus: role of endoscopic and histological predictors. Nat Rev Gastroenterol Infl ammatory and Non-Neoplastic Lesions Hepatol. 2014;11(9):525–534. Angulo-Pernett F, Smythe WR. Primary lymphoepithelioma of the Abe T, Hosokawa M, Kusumi T, et al. Adenocarcinoma arising from esophagus. Ann Thorac Surg. 2003;76(2):603–605. ectopic gastric mucosa in the cervical esophagus. Am J Clin Oncol. Appelman HD, Matejcic M, Parker MI, et al. Progression of esophageal 2004;27(6):644–645. dysplasia to cancer. Ann NY Acad Sci. 2014;1325(1):96–107. 172 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combina- Sampliner RE. Updated guidelines for the diagnosis, surveillance, and tion with chemotherapy versus chemotherapy alone for treatment therapy of Barrett’s esophagus. Am J Gastroenterol. 2002;97(8): of HER2-positive advanced gastric or gastro-oesophageal junction 1888–1895. cancer (ToGA): a phase 3, open-label, randomised controlled trial. Schnell TG, Sontag SJ, Chejfec G, et al. Long-term nonsurgical manage- Lancet. 2010;376(9742):687–697. ment of Barrett’s esophagus with high-grade dysplasia. Gastroenterol. Chen S, Chen Y, Yang J, et al. Primary mucoepidermoid carcinoma of 2001;120(7):1607–1619. the esophagus. J Thorac Oncol. 2011;6(8):1426–1431. Skacel M, Petras RE, Gramlich TL, Sigel JE, Richter JE, Goldblum Davies AR, Gossage JA, Zylstra J, et al. Tumor stage after neoadju- JR. The diagnosis of low-grade dysplasia in Barrett’s esophagus vant chemotherapy determines survival after surgery for adeno- and its implications for disease progression. Am J Gastroenterol. carcinoma of the esophagus and esophagogastric junction. J Clin 2000;95(12):3383–3387. Oncol. 2014;32(27):2983–2990. Spechler SJ, Souza RF. Barrett’s esophagus. N Engl J Med. Davydov CE, Delektorskaya VV, Kuvshinov YP, et al. Superficial and 2014;371(9):836–845. early cancers of the esophagus. Ann NY Acad Sci. 2014;1325(1): Verbeek RE, Leenders M, Ten Kate FJ, et al. Surveillance of Barrett’s esoph- 159–169. agus and mortality from esophageal adenocarcinoma: a population- Downs-Kelly E, Mendelin JE, Bennett AE, et al. Poor interobserver based cohort study. Am J Gastroenterol. 2014;109(8):1215–1222. agreement in the distinction of high grade dysplasia and adeno- Wainberg ZA, Anghel A, Desai AJ, et al. Lapatinib, a dual EGFR carcinoma in pretreatment Barrett’s esophagus biopsies. Am and HER2 kinase inhibitor, selectively inhibits HER2-amplified J Gastroenterol. 2008;103:2333–2340. human gastric cancer cells and is synergistic with trastuzumab in Estrella JS, Hofstetter WL, Correa AM, et al. Duplicated muscularis vitro and in vivo. Clin Cancer Res. 2010;16(5):1509–1519. mucosae invasion has similar risk of lymph node metastasis and Weston AP, Sharma P, Mathur S, et al. Risk stratification of Barrett’s recurrence-free survival as intramucosal esophageal adenocarci- esophagus: updated prospective multivariate analysis. Am J noma. Am J Surg Pathol. 2011;35(7):1045–1053. Gastroenterol. 2004;99(9):1657–1666. Flejou JF, Odze RD, Montgomery E, et al. Adenocarcinoma of the Yantiss RK. Diagnostic challenges in the pathologic evaluation of Barrett oesophagus. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, esophagus. Arch Pathol Lab Med. 2010;134(11):1589–1600. eds. WHO Classification of Tumours of the Digestive System. Zhu W, Appelman HD, Greenson JK, et al. A histologically defined 4th ed. Lyon: International Agency for Research on Cancer; subset of high-grade dysplasia in Barrett mucosa is predictive of 2010:25–31. associated carcinoma. Am J Clin Pathol. 2009;132(1):94–100. Hagen CE, Lauwers GY, Mino-Kenudson M. Barrett esophagus: diag- nostic challenges. Semin Diagn Pathol. 2014;31(2):100–113. Hermann RM, Horstmann O, Haller F, et al. Histomorphological Neuroendocrine Tumors tumor regression grading of esophageal carcinoma after neoad- juvant radiochemotherapy: which score to use? Dis Esophagus. Helle KB. Chromogranins A and B and secretogranin II as prohormones 2006;19(5):329–334. for regulatory peptides from the diffuse neuroendocrine system. Hobel S, Dautel P, Baumbach R, et al. Single center experience of endo- Results Probl Cell Differ. 2010;50:21–44. scopic submucosal dissection (ESD) in early Barrett s adenocarci- Hoang MP, Hobbs CM, Sobin LH, Albores-Saavedra J. Carcinoid noma. Surg Endosc. 2014. [Epub ahead of print] tumor of the esophagus: a clinicopathologic study of four cases. Leers JM, DeMeester SR, Oezcelik A, et al. The prevalence of lymph Am J Surg Pathol. 2002;26(4):517–522. node metastases in patients with T1 esophageal adenocarcinoma Pearse AG, Polak JM. Neural crest origin of the endocrine polypep- a retrospective review of esophagectomy specimens. Ann Surg. tide (APUD) cells of the gastrointestinal tract and pancreas. Gut. 2011;253(2):271–278. 1971;12(10):783–788. Lomo LC, Blount PL, Sanchez CA, et al. Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett’s esophagus cohort. Am J Surg Pathol. 2006;30(4):423–435. Mesenchymal Tumors Menezes A, Tierney A, Yang YX, et al. Adherence to the 2011 American Fei BY, Yang JM, Zhao ZS. Differential clinical and pathological char- Gastroenterological Association medical position statement for the acteristics of esophageal stromal tumors and leiomyomata. Dis diagnosis and management of Barrett’s esophagus. Dis Esophagus. Esophagus. 2014;27(1):30–35. 2014. [Epub ahead of print] Goldblum JR, Rice TW, Zuccaro G, Richter JE. Granular cell tumors Merlo LM, Shah NA, Li X, et al. A comprehensive survey of clonal of the esophagus: a clinical and pathologic study of 13 cases. Ann diversity measures in Barrett’s esophagus as biomarkers of progres- Thorac Surg. 1996;62(3):860–865. sion to esophageal adenocarcinoma. Cancer Prev Res. 2010;3(11): Miettinen M, Sarlomo-Rikala M, Sobin LH, Lasota J. Esophageal 1388–1397. stromal tumors: a clinicopathologic, immunohistochemical, Noble F, Nolan L, Bateman AC, et al. Refining pathological evalua- and molecular genetic study of 17 cases and comparison with tion of neoadjuvant therapy for adenocarcinoma of the esophagus. esophageal leiomyomas and leiomyosarcomas. Am J Surg Pathol. World J Gastroenterol. 2013;19(48):9282–9293. 2000;24(2):211–222. Okines A, Cunningham D, Chau I. Targeting the human EGFR family in esophagogastric cancer. Nat Rev Clin Oncol. 2011;8(8):492–503. Patil DT, Golblum JR, Rybicki L, et al. Prediction of adenocarcinoma Miscellaneous Other Tumors in esophagectomy specimens based upon analysis of preresection biopsies of Barrett esophagus with at least high trade dysplasia: a Bisceglia M, Perri F, Tucci A, et al. Primary malignant melanoma of the comparison of two systems. Am J Surg Pathol. 2012;36:134–141. esophagus: a clinicopathologic study of a case with comprehensive Sami SS, Subramanian V, Butt WM, et al. High definition versus stan- literature review. Adv Anat Pathol. 2011;18(3):235–252. dard definition white light endoscopy for detecting dysplasia in Chen ZM, Scudiere JR, Abraham SC, Montgomery E. Pyloric gland patients with Barrett’s esophagus. Dis Esophagus. 2014. [Epub adenoma: an entity distinct from gastric foveolar type adenoma. ahead of print] Am J Surg Pathol. 2009;33(2):186–193. 8 Neoplasms of the Stomach LAUR A W. LAMPS AND SCOT T R . OW ENS INTRODUCTION Infl ammatory Polyps There are no well-defined criteria for inflammatory polyps A wide variety of benign and malignant neoplasms arise in the stomach. Some pathologists use this term to refer to in the stomach; they can be broadly classified as epithe- polypoid granulation tissue, or synonymously with “pol- lial, neuroendocrine, mesenchymal, and hematolymphoid ypoid gastritis,” to reflect marked chronic gastritis that (Table 8.1). Gastric adenocarcinoma remains the second produces nodular or polypoid inflammatory lesions. This most common malignancy worldwide, although there has most often happens in the context of Helicobacter pylori been a decline in its incidence over the past five decades. infection, although it has also been described in associa- Well differentiated neuroendocrine tumors (NETs) have tion with chronic atrophic gastritis. comprised less than 1% of gastric neoplasms in the past; this number is likely a significant underestimate, and has increased in recent years due to awareness of these types of Hyperplastic Polyps tumors and improved imaging and endoscopic techniques. Hyperplastic polyps are reportedly the second most com- Although mesenchymal tumors reportedly account for less mon gastric polyp after fundic gland polyps (FGPs), than 1.0% of gastrointestinal (GI) malignancies overall, although diagnostic criteria have not been clearly defined most of these are gastrointestinal stromal tumors (GISTs), in the past (see discussion in subsequent paragraphs). many of which (60%–70%) arise in

the stomach. Gastric Most are discovered incidentally during upper endos- lymphoma accounts for approximately 5% to 10% of gas- copy for another reason. They have been associated tric malignancies. This chapter will address the most com- with autoimmune gastritis and with longstanding H. monly encountered tumors of the stomach; neoplasms that pylori-associated gastritis, as well as other inflamma- can affect any part of the GI tract, but infrequently affect tory conditions. Historically, entities termed hyperplas- the stomach, are discussed elsewhere. tic polyp have represented a very heterogeneous group of polyps that also include inflammatory polyps, foveo- lar hyperplasia, and gastric mucosal prolapse polyps. INFLAMMATORY AND Numerous hyperplastic polyps may raise the possibility NON-NEOPLASTIC LESIONS of gastric hyperplastic polyposis, although neither the clinical nor the pathologic criteria for this rare disorder Gastroenterologists commonly biopsy inflammatory or have been well elucidated. non-neoplastic gastric lesions and polyps that are found More recent criteria suggest classifying lesions con- incidentally at endoscopy. Some of these mimic or enter sisting of a sharply localized focus of elongated foveolar into the differential diagnosis of other gastric neoplasms, glands without cystic changes, significant inflammation, or have an associated risk of developing dysplasia, and or architectural derangement as “polypoid foveolar hyper- thus will be discussed here briefly. plasia” (Figure 8.1A–B). Hyperplastic polyps, in contrast, 173 174 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 8.1 Classification of Gastric Neoplasms According to the 2010 World Health Organization Epithelial Tumors Premalignant Lesions Adenoma Intraepithelial neoplasia, low grade Intraepithelial neoplasia, high grade Carcinoma Adenocarcinoma Papillary Tubular Mucinous Poorly cohesive (including signet ring cell carcinoma) Mixed Adenosquamous carcinoma Medullary carcinoma Hepatoid adenocarcinoma Squamous cell carcinoma Undifferentiated carcinoma Neuroendocrine Neoplasms Neuroendocrine tumor (NET) G1 (carcinoid) G2 (A) Neuroendocrine carcinoma (NEC) Large cell NEC Small cell NEC Mixed adenoneuroendocrine tumor (MANEC) Enterochromaffin (EC) cell, serotonin producing NET Gastrinoma (gastrin-producing NET) Mesenchymal Tumors Glomus tumor Granular cell tumor Leiomyoma Plexiform fibromyxoma Inflammatory myofibroblastic tumor Gastrointestinal stromal tumors (GIST) Kaposi sarcoma Leiomyosarcoma Synovial sarcoma Lymphoma Secondary Tumors Source: Data adapted from WHO Classification of Tumors of the Digestive (B) System, 4th Edition, 2010. FIGURE 8.1 Polypoid foveolar hyperplasia features elongated, tortuous foveolar glands without cystic changes or significant inflammation (A–B). Mucin feature elongation of the pit region imparting a corkscrew depletion is common in the epithelium. appearance, branching and cystic dilatation of glands, lamina propria edema and inflammation, and variably present surface erosions (Figure 8.2A–C). Pseudogoblet cells, or distended mucinous cells, are frequently seen as or nonspecific chronic inactive gastritis. Because the well (Figure 8.2D). Although there may be thin strands of majority of hyperplastic polyps are associated with some smooth muscle, the thick bundles of muscle typically seen form of gastritis, biopsy of intervening nonpolypoid in hamartomatous polyps are not a feature of hyperplastic mucosa is strongly recommended. Many hyperplastic pol- polyps. Hyperplastic polyps can be pedunculated or ses- yps regress with treatment of the background gastritis, but sile, and range in size from a few millimeters to 3 cm or recurrence is common. more. They are most common in the antrum, but can be Hyperplastic polyps often contain foci of intestinal found anywhere in the stomach, and are often multiple. metaplasia, but intestinal metaplasia is even more likely Hyperplastic polyps and polypoid foveolar hyperplasia to be found in the mucosa adjacent to hyperplastic polyps. may well represent related lesions along a spectrum of There is an associated very low (approximately 4%) but regenerative/reactive polyps, and both lesions can be seen well-recognized risk of dysplasia, especially when hyper- in the context of reactive gastropathy, H. pylori infection, plastic polyps are large (over 2 cm) (Figure 8.2E–F). 8 Neoplasms of the Stomach 175 Gastric Mucosal Prolapse Polyps (Figure 8.3A–B). A prominent basal glandular compo- nent consisting of tightly packed, back-to-back glands is Gastric mucosal prolapse polyps are a more recently another distinguishing feature. described inflammatory lesion typically seen in the antrum and pylorus of middle-aged patients. They may be sessile, Heterotopic Pancreas/Adenomyoma papular, or pedunculated, and range in size from a few millimeters to over 3 cm. Similar to hyperplastic polyps, Pancreatic rests or pancreatic heterotopias are congenital mucosal prolapse polyps show elongation and cystic dila- lesions that represent separation of portions of pancreatic tation of the gastric pits, but also feature bundles of arbo- tissue during rotation of the foregut. They present in a vari- rizing smooth muscle and thick-walled, prominent vessels ety of ways, ranging from asymptomatic incidental findings (A) (B) (C) (D) FIGURE 8.2 Hyperplastic polyps feature elongation of the pit region (A [courtesy Dr. Rhonda Yantiss] and B), imparting a corkscrew appearance, along with branching and cystic dilatation of glands and lamina propria edema and inflammation (C). Pseudogoblet cells are frequently seen as well (D, courtesy Dr. Rhonda Yantiss). (continued) 176 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (E) (F) FIGURE 8.2 (continued) There is a low but well-recognized risk of dysplasia in hyperplastic polyps, especially in those greater than 2 cm (E, low-grade dysplasia [courtesy Dr. Rhonda Yantiss]; F [arrow], high grade dysplasia/carcinoma in situ). (A) (B) FIGURE 8.3 Mucosal prolapse polyps show elongation and variable dilatation of the gastric pits, as well as prominent bundles of arborizing smooth muscle and prominent vessels (A–B). A prominent basal glandular component consisting of tightly packed, back-to-back glands is another distinguishing feature. 8 Neoplasms of the Stomach 177 to ulceration with associated GI b leeding. The c lassic endo- Histologically, pancreatic heterotopia consists of scopic appearance is that of a bulging d ome-shaped mass irregularly dilated cystic glands lined by cuboidal epi- with a broad base, smooth surface, and central umbilication thelium, resembling biliary epithelium. These ductal (Figure 8.4A). In the stomach, pancreatic heterotopias are structures are arranged in a lobular pattern, with sur- most often located in the antrum. rounding dense fibromuscular stroma and (usually) Although usually asymptomatic, pancreatic hetero- pancreatic exocrine and endocrine cells, including islets topias have several clinically important sequelae. They of Langerhans (Figure 8.4B–C). Occasionally, however, may present with localized inflammation and ulceration only ductal epithelium is present (sometimes called an that leads to bleeding, and more rarely, they cause pyloric “adenomyoma”), which may mimic invasive adenocarci- obstruction or intussusception due to mass effect. Rare noma, particularly on frozen section. In addition, lesions cases of malignant transformation (both adenocarcinoma with prominent islet cells can occasionally mimic NET in and well- differentiated NETs) have been reported. a biopsy specimen. (A) (B) (C) FIGURE 8.4 Macroscopically, pancreatic heterotopia often appears as an umbilicated nodule (A). The lesion is composed of ducts arranged in a lobular pattern (B), with surrounding dense fibromuscular stroma and pancreatic exocrine and endocrine cells, including islets of Langerhans (C). 178 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Diagnostic Challenges syndrome (PJS), juvenile polyposis syndromes (JPS), and Cowden disease; these are discussed in more detail in Hyperplastic polyps and inflammatory-type polyps pres- Chapter 6. ent two distinct diagnostic challenges in terms of dif- ferential diagnosis. The first is differentiating between a hyperplastic or inflammatory lesion with marked Fundic Gland Polyps regenerative/reparative changes, and dysplasia. Nuclear Fundic gland polyps (FGPs) are the most common hamar- enlargement and hyperchromasia, prominent nucleoli, tomatous gastric polyp, and one of the most common types and increased mitotic activity are all features of reparative of gastric polyp overall. These polyps may be syndromic atypia that can mimic dysplasia. Most reparative changes (most commonly associated with familial adenomatous that are severe enough to mimic dysplasia are also asso- polyposis [FAP]) or sporadic. FGPs are also associated ciated with marked acute inflammation and/or mucosal with Zollinger–Ellison syndrome and with the rare entity ulceration, and therefore cytologic atypia in association known as sporadic fundic gland polyposis, defined as with marked inflammation and/or ulceration should be multiple fundic gland polyps in patients without FAP or interpreted with caution. It is also important to remember related syndromes. FGPs in sporadic fundic gland polypo- that immunostains such as p53 may be positive in areas sis are histologically identical to solitary sporadic FGP, of marked regeneration as well. Inflammatory changes but demonstrate frequent somatic activating mutations in tend to be uniform and show a gradual transition to more exon 3 of CTNNB1, which encodes β-catenin. normal-appearing mucosa, whereas dysplasia is sharply FGPs are smooth, sessile lesions that by definition demarcated from the adjacent non-neoplastic mucosa. occur in a background of gastric oxyntic mucosa. They The second challenge is distinguishing between hyper- may be single or multiple, and are often multiple in the plastic polyps and other entities in the differential diag- context of FAP. Typically these are small lesions (less than nosis, including hamartomatous polyps and Ménétrier 1.0 cm), but may grow to 2 cm to 3 cm. Histologically, disease. Gastric Peutz–Jeghers polyps (PJPs) typically have FGPs are composed of oxyntic as well as foveolar epithe- well-developed lobular architecture, and lack the cysti- lium (Figure 8.5A–B). There is frequently cystic dilata- cally dilated, tortuous glands and pits as well as the edem- tion of glands, as well as prominent epithelial budding. atous, inflamed stroma typical of hyperplastic polyps. Inflammation is not prominent. The overlying surface Hyperplastic polyps also typically lack the well-developed foveolae are shortened. Patients on protein pump inhibi- smooth muscle component often seen in Peutz–Jeghers tor (PPI) therapy may show parietal cell hypertrophy and polyps. Gastric hyperplastic polyps and gastric juvenile hyperplasia, with tufting of cells into the lumen as well as polyps can have substantial histologic overlap, and thus vacuolization of the cytoplasm (Figure 8.5C). clinical and family history as well as endoscopic informa- Sporadic FGPs are increasing in incidence, which tion may be necessary to distinguish these lesions. Gastric is most likely a reflection of increasing PPI use as these hyperplastic polyps can also mimic Ménétrier disease and drugs have been implicated in the pathogenesis of spo- Cronkhite–Canada syndrome, particularly if only a small radic FGPs. Women are more likely to develop sporadic biopsy of a larger endoscopic lesion is available for evalu- FGPs, whereas syndromic FGPs show no gender predilec- ation. Knowledge of the clinical history and endoscopic tion. Sporadic FGPs have essentially no malignant poten- findings is essential in this circumstance as well. Gastritis tial, and dysplasia occurs in less than 1.0% of sporadic cystica profunda/polyposis may also mimic hyperplastic lesions. polyps. This condition typically develops in patients who Approximately 90% of patients with FAP have FGPs, are status-post surgery, particularly if bile reflux is pres- which frequently show dysplasia (Figure 8.6A–B). In this ent. If the lesion is polypoid and intraluminal, then the context, dysplasia is associated with larger polyp size, polyposa moniker is used; if the lesions are intramural, antral gastritis, and increased severity of duodenal pol- then profunda is used. Patients develop polypoid lesions yposis; PPI therapy appears to be protective against the that are similar to hyperplastic polyps or prolapse pol- development of dysplasia. The dysplasia is typically low- yps; in addition, as in colitis cystica profunda, entrapped grade and focal, although occasional polyps show more benign gastric epithelium is often present beneath the extensive abnormalities or high-grade dysplasia. The bio- mucosal surface. logic risk of developing gastric adenocarcinoma from these lesions is quite low, and thus patients are usually monitored with surveillance endoscopy and regular removal of large EPITHELIAL NEOPLASMS lesions without prophylactic gastrectomy. Pathologists may suggest the possibility of a polyposis disorder when FGPs Hamartomatous Polyps with dysplasia are encountered, particularly when multi- Several types of hamartomatous polyps occur in the ple, or if FGPs are detected in children who have not been stomach, including polyps associated with Peutz–Jeghers exposed to PPIs. 8 Neoplasms of the Stomach 179 (A) (B) (C) FIGURE 8.5 Fundic gland polyps are composed of a mixture of oxyntic as well as foveolar epithelium (A–B). There is frequently cystic dilatation of glands, and inflammation is not prominent. Fundic gland polyps from patients on PPIs may show parietal cell hypertrophy and hyperplasia, with tufting of cells into the lumen and vacuolization of the cytoplasm (C). Peutz–Jeghers Syndrome As noted in the preceding paragraphs, gastric PJPs are very similar histologically to hyperplastic polyps, Peutz-Jeghers syndrome (PJS) is an inherited syndrome and the only distinguishing features may be a more characterized by a germline mutation of the tumor sup- prominent smooth muscle component and less promi- pressor gene STK11/LKB1. Patients typically have polyps nent cystically dilated glands. Gastric PJPs also contain throughout the GI tract, as well as mucocutaneous mela-

normal-appearing lamina propria, rather than the edem- nocytic pigmentation. The polyps preferentially affect atous, inflamed stroma typical of hyperplastic polyps the small intestine, but are seen in the stomach in 25% and juvenile polyps. In many cases, however, histologic to 50% of patients. Gastric PJPs are typically small and distinction from hyperplastic polyps is impossible, and asymptomatic, and macroscopically similar to PJ polyps the diagnosis rests on the knowledge that the patient has elsewhere in the GI tract with a villiform or papillary sur- PJS. Patients with McCune–Albright syndrome may also face. Histologically, PJ polyps show irregular arborizing have polyps that are identical to PJPs. bundles of smooth muscle that extend upward from the Although some literature cites the development of gas- muscularis mucosae to separate the epithelial components tric dysplasia and adenocarcinoma in PJS as rare, more into lobules (Figure 8.7A–B). Surface and foveolar hyper- recent studies indicate a 29% lifetime risk of developing plasia is common, along with cystic dilatation of glands. gastric cancer. It is recommended that patients with PJS 18 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 8.6 These fundic gland polyps from patients with familial adenomatous polyposis show low-grade dysplasia at the surface of the polyp (A [courtesy Dr. Rhonda Yantiss]–B). begin surveillance of the upper and lower GI tracts at Juvenile Polyps age 8; if no polyps are seen, then re-evaluation is recom- Juvenile polyps may be either sporadic or syndromic, mended at age 18 and every 3 years thereafter. If polyps but sporadic polyps are very rare in the stomach. Gastric are seen, evaluation every 3 years is recommended. (A) (B) FIGURE 8.7 Peutz–Jeghers polyps are composed of irregular arborizing bundles of smooth muscle that extend upward from the muscularis mucosae to separate the epithelial components into lobules (A). Surface and foveolar hyperplasia is common, along with cystic dilatation of glands (B). 8 Neoplasms of the Stomach 181 juvenile polyps typically occur as part of generalized JPS typically presents in middle-aged adults. Patients with or the rare subtype that is limited to the stomach. JPS is CCS have numerous GI polyps, as well as diarrhea, characterized by a germline mutation in the SMAD4 or weight loss, anorexia, abdominal pain, and GI bleeding. BMPR1A gene, and 20% to 50% of patients with JPS Extraintestinal manifestations include skin hyperpigmen- have gastric polyps. In addition to the increased risk of tation, vitiligo, alopecia, and nail atrophy. The mortality developing colorectal cancer, JPS patients are also at risk rate is quite high (approximately 50%), most often due to for small intestinal, gastric, and possibly pancreatic can- anemia and chronic malnutrition. In addition, patients cer. Surveillance recommendations for JPS include begin- have an increased risk of adenocarcinoma in the stomach ning upper endoscopy and colonoscopy at ages 12 to 15, as well as other parts of the GI tract. The increased risk and repeating every 1 to 3 years depending on symptoms of malignancy is not directly related to the presence of the and findings. polyps, apparently, as cancers may develop in the polyps Gastric JP range from a few millimeters to several or in nonpolypoid mucosa. centimeters in size, and histologically feature cystic dila- In the stomach, CCS features diffuse but irregular tation of glands within an edematous, inflamed stroma. hypertrophy of the gastric rugae. Polyps typically mea- Foveolar hyperplasia and smooth muscle hyperplasia may sure between 5 and 15 mm, and are superimposed on the also be present (Figure 8.8A). Gastric JP can mimic hyper- hypertrophic rugae. A very helpful feature of CCS is that plastic polyps or Ménétrier disease, particularly in biopsy the histologic changes involve both the polypoid and non- samples. Because of the histologic overlap with hyperplas- polypoid mucosa, and include marked surface and foveo- tic polyps and other hamartomatous polyps, knowledge of lar hyperplasia with cystic dilatation as well as atrophy the clinical history is essential. Syndromic gastric juvenile of glands (Figure 8.9A–B). The lamina propria is edema- polyps may also develop dysplasia (Figure 8.8B), and thus tous and inflamed, often with prominent eosinophils, but patients are at increased risk for the development of gas- intestinal metaplasia is not a typical feature. The nonpol- tric adenocarcinoma as well. ypoid mucosa shows similar alternating areas of hyper- plasia and atrophy with cystic changes, which can help Cronkhite–Canada Syndrome differentiate CCS from histologically similar lesions such as juvenile polyps, in which the nonpolypoid mucosa is Cronkhite–Canada Syndrome (CCS) is a very rare, non- typically normal. The microscopic differential diagnosis hereditary generalized polyposis disorder that involves also includes Ménétrier disease, hyperplastic polyps, and the stomach, small bowel, and colorectum. The patho- other hamartomatous polyps, and thus knowledge of the genesis remains unclear, although an autoimmune etiol- clinical history is essential. In contrast to Ménétrier dis- ogy has been proposed. Unlike most polyposis syndromes, ease, CCS involves the entire stomach and is not limited to which present in childhood or young adulthood, CCS the body and fundus. (A) (B) FIGURE 8.8 Gastric juvenile polyps feature cystic dilatation of glands within edematous, inflamed stroma (A). Syndromic gastric juvenile polyps may also develop dysplasia, as seen here at the surface of a large juvenile polyp (B, arrows); note the adjacent cystically dilated glands and inflamed, edematous stroma. 18 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 8.9 The polyps in gastric Cronkhite–Canada syndrome show marked surface and foveolar hyperplasia with cystic dilatation as well as atrophy of glands, similar to hyperplastic polyps (A). The lamina propria is edematous and mildly inflamed. The nonpolypoid mucosa shows similar alternating areas of hyperplasia and atrophy with cystic changes (B). Courtesy Dr. Rhonda Yantiss. GASTRIC DYSPLASIA Gastric dysplasia is morphologically classified as either adenomatous/intestinal or foveolar/gastric, based Gastric epithelial dysplasia is widely accepted as an impor- on histologic characteristics. The adenomatous/intestinal tant precursor lesion to gastric adenocarcinoma, as well as type resembles intestinal-type colonic adenomas, with a marker of increased risk of synchronous adenocarcinoma columnar cells containing hyperchromatic, pencillate, elsewhere in the stomach. The prevalence varies widely pseudostratified nuclei (Figure 8.10A–D). In contrast, the depending on population, with rates ranging between 0.5 foveolar type consists of cuboidal to columnar cells with and 3.75% in Western countries, but much higher (rang- clear to eosinophilic cytoplasm and hyperchromatic round ing from 9%–20%) in high-risk geographic areas such as to ovoid nuclei (Figure 8.11A–B). Some gastric dysplasias Korea, China, Japan, and Colombia. Historically, polyp- consist of a mixture of both types. Either type can be asso- oid dysplasias are referred to as adenomas in the United ciated with intestinal metaplasia, as well as the develop- States and Europe, whereas flat, sessile, or depressed ment of high-grade dysplasia and adenocarcinoma; the lesions are more often referred to as flat or nonpolypoid rates of development of high-grade dysplasia and/or car- dysplasia, or simply dysplasia. In Japan, however, the term cinoma vary widely in the literature, most likely based on “adenoma” includes all gross types of dysplasia including the patient population studied, the presence of H. pylori polypoid, flat, or depressed lesions. Regardless of configu- infection, and genetic predisposition. Adenomas them- ration, the histologic features are essentially identical, and selves (and flat dysplasia) are almost always asymptomatic therefore some authors recommend grouping all of these and found incidentally on endoscopy. However, as they dysplastic lesions into the term “gastric intra-epithelial often occur in a background of chronic gastritis, patients neoplasia/dysplasia.” may present with melena, abdominal pain, nausea and Risk factors for the development of gastric epithelial vomiting, or anemia. dysplasia include, most prominently, H. pylori infection Macroscopically, adenomas and flat dysplasia can be (particularly if intestinal metaplasia/atrophy is present), located anywhere in the stomach. Polypoid adenomas are and atrophic gastritis. Cigarette smoking, low serum vita- most commonly found in the antrum, and the vast major- min C levels, high salt consumption, and malnutrition ity are solitary. Most measure less than 2.0 cm, and grossly have been implicated as risk factors as well. Gastric ade- they may have either a pedunculated, nodular, or sessile nomas are also found in up to 15% of patients with FAP. configuration. Flat or depressed dysplastic foci may be 8 Neoplasms of the Stomach 18 3 (A) (B) (C) (D) FIGURE 8.10 The adenomatous or intestinal type of gastric dysplasia resembles colonic adenomas, and is composed of columnar cells containing hyperchromatic, pencillate, pseudostratified nuclei (A–B). Neuroendocrine cells are common (C), as are goblet cells (D). very difficult to detect with conventional endoscopy, but features more complex architecture (such as cribriform- may have an irregular appearance on chromoendoscopy. ing or budding of glands) and high-grade nuclear fea- As discussed in the previous paragraphs, gastric intes- tures with prominent nucleoli and loss of nuclear polarity tinal type dysplasia resembles a colonic adenoma, and this (Figure 8.12A–C). However, classification of dysplasia type of dysplasia comprises the majority of dysplastic lesions differs in other parts of the world, particularly Japan. The in the stomach. Goblet cells, Paneth cells, and neuroendo- most significant difference between Western and Japanese crine cells are often seen (Figure 8.10C–D). Foveolar dys- classifications is that Western pathologists require lamina plasia (Figure 8.11A–B), in contrast, lacks goblet and Paneth propria invasion to establish a diagnosis of intramucosal cells, and is composed of round or cuboidal to columnar adenocarcinoma, whereas Japanese pathologists place cells with clear to eosinophilic cytoplasm and hyperchro- greater emphasis on cytologic features of malignancy. For matic round to oval nuclei. these reasons, cases diagnosed as high-grade dysplasia in Similar to colonic adenomas, gastric dysplasia (both Western countries may well be classified as adenocarci- intestinal and foveolar types) is classified as either low or noma by Japanese pathologists. There are also multiple high grade in Western countries. High-grade dysplasia international grading schemes for the classification of 18 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 8.11 Foveolar-type gastric dysplasia consists of cuboidal to columnar cells with clear to eosinophilic cytoplasm and hyperchromatic, round to oval nuclei (A–B). Neuroendocrine cells and goblet cells are typically absent. (A) (B) (C) FIGURE 8.12 High-grade dysplasia features more complex architecture (A) and high-grade nuclear features with prominent nucleoli, loss of nuclear polarity, and nuclear stratification extending to the luminal surface (B–C). 8 Neoplasms of the Stomach 18 5 gastric dysplasia; the Vienna classification is the most (due to size, location, etc.) should be managed surgically. widely used in the United States. Eradication of H. pylori in patients with gastric dysplasia The progression from dysplasia to carcinoma is vari- as well as careful clinical follow-up with regular endos- able, but reportedly over 20% of low-grade lesions prog- copy and biopsy is recommended due to the risk of devel- ress to carcinoma within 10 to 48 months, and up to 85% oping subsequent lesions. of high-grade lesions progress over a similar period. The risk of neoplastic transformation of adenomas increases Differential Diagnosis with size, particularly once the lesion exceeds 2.0 cm. and Diagnostic Challenges Because of the high risk of development of adenocarci- noma, all dysplastic lesions should be completely removed The most problematic issue in the differential diagnosis and submitted for pathologic evaluation. In addition, the of gastric dysplasia is differentiation from marked regen- remainder of the stomach should be carefully evaluated erative/reparative atypia. Reactive changes may be partic- and biopsied for additional lesions, particularly flat dys- ularly worrisome in the context of chronic bile reflux in plasia. First-line treatment options include endoscopic a postoperative stomach, as these patients are at risk for polypectomy for pedunculated polyps, and endoscopic gastric dysplasia/carcinoma. Caution should be exercised mucosal resection (EMR) or endoscopic submucosal in this situation, as the reactive changes induced by bile dissection (ESD) for large and/or sessile polyps. Lesions reflux can be very striking (Figure 8.13A–C). True dyspla- that are not amenable to polypectomy or local excision sia should extend to the surface of the gastric mucosa in (A) (B) (C) FIGURE 8.13 Reactive gastric epithelium may show marked nuclear enlargement and hyperchromasia, prominent nucleoli, and increased mitotic activity that may mimic dysplasia, as shown here in a case of erosive gastritis (A, arrow denotes focus of muciphages; courtesy Dr. Rhonda Yantiss). Similar nuclear changes are seen in this case of bile reflux (B). The changes are associated with mucosal erosion, however, and do not extend to the surface (C). In addition, dysplasia is sharply demarcated from the adjacent

non-neoplastic mucosa, whereas reactive changes show a more gradual transition to normal mucosa. 18 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide both intestinal- and foveolar-type dysplasia. Furthermore, for dysplasia.” Similar issues may arise in biopsies of gas- most reparative changes that are severe enough to mimic tric ulcer beds when marked inflammatory and regenera- dysplasia are also associated with marked acute inflamma- tive changes, including exuberant granulation t issue, mimic tion and/or mucosal ulceration. Reactive changes tend to dysplasia and neoplasia. be uniform and show a gradual transition to more normal- Chemotherapeutic agents and radiation, including appearing mucosa, whereas dysplasia is sharply demarcated gastric injury secondary to microsphere radioemboliza- from the adjacent non-neoplastic mucosa. It is important tion, can also produce severe epithelial abnormalities that to remember, as in other parts of the GI tract, that immu- mimic dysplasia (Figure 8.14A–C). Although these changes nostains such as p53 may be positive in areas of marked include nuclear enlargement and hyperchromasia with regeneration as well as foci of dysplasia. Epithelial lesions membrane irregularities, affected cells often contain vacu- worrisome for dysplasia, but for which the diagnosis cannot olated cytoplasm, and although there is nuclear enlarge- be made with certainty, may be interpreted as “indefinite ment the nuclear-to-cytoplasmic ratio is maintained. (A) (B) (C) FIGURE 8.14 This biopsy from a patient receiving chemotherapy shows nuclear enlargement, bizarre nuclear pleomorphism, and loss of nuclear polarity. However, the affected cells have vacuolated cytoplasm, and the nuclear-to- cytoplasmic ratio is maintained (A). Similar changes are seen in this biopsy from a patient who had undergone radiation therapy (B, courtesy Dr. Rhonda Yantiss). Gastric injury secondary to microsphere radioembolization can also produce severe reactive abnormalities that mimic dysplasia (C, courtesy Dr. Shawn Kinsey). 8 Neoplasms of the Stomach 187 Other entities in the differential diagnosis of gastric than 3% of all gastric polyps, reportedly, but are likely adenomas include FGPs, hyperplastic polyps, or other underrecognized. Gastric PGAs tend to arise in elderly types of hamartomatous polyps with dysplasia. The back- patients and are more common in women. Most arise in ground changes in these lesions typically help distinguish the body, but they may occur anywhere in the stomach. them from true gastric adenomas. In addition, the dyspla- The surrounding gastric mucosa usually shows evidence sia in these other types of polyps is typically focal. Finally, of autoimmune gastritis or H. pylori gastritis. dysplasia is unusual in FGPs and other types of hamar- PGAs are often relatively large at the time of diag- tomatous polyps that are not associated with a polyposis nosis, with a mean diameter of 1.6 cm. They are com- syndrome. posed of closely packed pyloric-type tubules or glands lined by a single layer of columnar or cuboidal epithe- lium (Figure 8.15A–B). The cytoplasm is eosinophilic or OTHER EPITHELIAL POLYPS amphophilic, and often has a “ground-glass” appearance. Apical mucin caps are absent. Nuclei are characteristically Pyloric Gland Adenomas round, and may or may not contain easily identifiable Pyloric gland adenoma (PGA) is a rare type of adenoma nucleoli. By immunohistochemistry, PGAs characteristi- that most often involves the stomach; they account for less cally express MUC6 (a marker of pyloric gland mucin) (A) (B) (C) FIGURE 8.15 Pyloric gland adenomas are composed of closely packed pyloric-type tubules or glands (A) lined by a single layer of columnar or cuboidal epithelium. The cytoplasm is eosinophilic or amphophilic, and often has a “ground- glass” appearance. Apical mucin caps are absent. Nuclei are characteristically round, and may or may not contain easily identifiable nucleoli (B). Dysplasia is a common finding in these lesions (C, courtesy Dr. Rhonda Yantiss). 18 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide and MUC5AC (a foveolar mucin marker). The intestinal of cells, which can have notable nuclear hyperchromasia, mucin MUC2 and the intestinal marker CDX2 are gener- pleomorphism, anisocytosis, and enlargement. MUC6 is ally not expressed in PGAs. characteristically positive, whereas MUC5AC and MUC2 The term “pyloric gland adenoma” has been criticized, are negative. Oxyntic gland polyps lack the cystic dilata- because the name “adenoma” is used to describe a lesion tion and admixed foveolar cells typically seen in FGPs, that does not a priori have conventional-type dysplasia. and FGPs lack the nuclear atypia seen in oxyntic gland However, dysplasia is quite common in these lesions polyps. (Figure 8.15C). Dysplasia (both low and high grades) has been reported in up to 40% of PGAs, and there has also been a reported frequency of adenocarcinoma in up to ADENOCARCINOMA 30%. Little is known regarding prognosis, due to limited clinical and follow-up data. However, complete removal Epidemiology with subsequent close clinical follow-up and endoscopic Gastric adenocarcinoma accounts for approximately surveillance is recommended. 7% to 10% of cancers and is the second most common type of cancer worldwide; however, there is a striking geographical heterogeneity due to a variety of genetic Differential Diagnosis and environmental factors. This heterogeneity is fur- PGAs are likely underrecognized as they are often confused ther underscored by the wide spectrum of macroscopic with other types of polyps, particularly hyperplastic pol- and histologic findings in these tumors. High-incidence yps. PGAs lack the marked elongation, branching, and tor- areas include eastern Asia, central and eastern Europe, tuosity of the glands seen in hyperplastic polyps, and also Latin America, and South America, and antral and typically lack the inflammatory stroma. In addition, PGAs pyloric tumors are more common in these regions. are composed of pyloric-type glands rather than foveolar- Low-incidence areas include North America, northern type cells, and lack the apical mucin droplet characteristic Europe, most of Africa, and southeast Asia, and proxi- of foveolar epithelium. MUC6 should be absent in foveo- mal stomach/gastroesophageal (GE) junction tumors are lar epithelium, as well. Occasionally, foveolar-type gastric more common in these areas. The diffuse type of gas- adenomas may be confused with PGAs. As discussed in the tric carcinoma does not vary with region, and is more preceding section on foveolar-type dysplasia, these also are common in younger patients. composed of cells resembling foveolar epithelium, with Of note, there has been a declining overall incidence of different cytologic features and by definition at least low- gastric carcinoma over the past five decades, although there grade cytologic dysplasia. has been an increase in proximal and GE junction carcino- mas since the 1980s that parallels the increase in esopha- geal adenocarcinoma. This increase appears restricted to Oxyntic Gland Polyp/Adenoma low-incidence areas, and has not been observed in areas that are at high risk for the development of gastric carci- Nodular mucosa-based lesions composed of oxyntic noma, such as Japan. The reason for the increase is contro- mucosa have had several names, including chief cell hyper- versial, in part because of the lack of consensus as to the plasia, oxyntic mucosa pseudopolyp, and oxyntic gland definition of the gastric cardia, and in part because wide- polyp/adenoma, reflecting the confusion as to whether or spread use of endoscopy for surveillance has most likely not these represent mucosal hyperplasia or true neoplasia. resulted in increased detection of these tumors. Regardless, Some cases have also been termed adenocarcinoma with there are clear differences between gastric cardia carci- chief cell differentiation, although this term should prob- nomas and those in the more distal stomach, including a ably be avoided given the apparent lack of recurrence of greater incidence in men and Caucasians. Whether or not metastasis of these lesions, as well as the lack of associ- the risk factors associated with esophageal carcinoma (obe- ated desmoplasia, perineural invasion, and lymphovascu- sity, reflux, smoking, alcohol use) are also implicated in the lar invasion. pathogenesis of gastric cardiac cancers remains controver- These lesions are composed of a mixture of pari- sial, as does the significance of gastric cardiac intestinal etal and chief cells (Figure 8.16A–C), although some are metaplasia, and thus patients with intestinal metaplasia in almost exclusively composed of chief cells; they are most the gastric cardia do not undergo rigorous surveillance as often solitary and found in the body and fundus of the do patients with Barrett’s esophagus. stomach. Admixed mucus neck cells may also be pres- More than 80% of cases of gastric carcinoma are spo- ent, and may be quite prominent. They are centered in the radic, and appear to develop via a progression from chronic mucosa, and do not extend into the submucosa. The cells gastritis to atrophic gastritis to intestinal metaplasia, dys- are arranged in tightly packed clusters, tubules, and cords plasia, and carcinoma (the Correa cascade). A number of 8 Neoplasms of the Stomach 18 9 (A) (B) (C) FIGURE 8.16 This oxyntic gland adenoma is composed of a mixture of parietal and chief cells (A). The cells are arranged in tightly packed tubules (B), and have notable nuclear hyperchromasia, pleomorphism, anisocytosis, and enlargement (C). Courtesy Dr. Elizabeth Montgomery. risk factors, associated diseases, and precancerous condi- Clinical Features tions are associated with gastric cancer (particularly the Gastric carcinomas are typically divided into two general intestinal type), including chronic H. pylori infection, categories: early gastric cancer (EGC) and advanced gas- autoimmune gastritis, Ménétrier disease, and long-stand- tric cancer (AGC). EGC is defined as carcinoma limited to ing chemical gastropathy/bile reflux, especially in patients the mucosa or submucosa, regardless of lymph node status, who have undergone distal gastrectomy and a Billroth II whereas AGC is defined as a lesion that invades beyond procedure. High salt diets and nutrient-poor diets have the submucosa. EGC accounts for 15% to 20% of gastric been implicated, as they are believed to lead to the for- carcinomas in Western countries, but more than half of mation of intraluminal carcinogens such as N-nitroso cases in Japan, in large part due to the use of aggressive compounds. The diffuse type of gastric cancer is associ- surveillance and different diagnostic definitions of carci- ated more strongly with genetic abnormalities than envi- noma in high-risk areas (see preceding section on gastric ronmental factors, however (see subsequent paragraphs). dysplasia). EGCs are most often seen in male patients A minority of gastric cancers have a well-defined genetic over the age of 50, the majority of whom are asymptom- abnormality; these are also discussed subsequently. atic. When symptomatic, patients typically present with 19 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 8.2 Endoscopic Classification of Early Gastric Cancers Appearance Significance Additional Macroscopic Features Type I Protruding Low rate of lymph node metastases Type II Superficial Most common, especially IIa Type IIa Elevated Low rate of lymph node metastases Twice as thick as adjacent normal mucosa; hard to detect endoscopically Type IIb Flat Majority of tumors that are smaller than 0.5 cm Type IIc Depressed More likely to be poorly differentiated Mimics benign gastric ulcer Type III Excavating More likely to be poorly differentiated dyspepsia, epigastric pain, anemia, or melena. AGCs are 10 cm at presentation. There are a variety of macroscopic also more common in men over the age of 50. Symptoms appearances, including a polypoid or exophytic mass; include epigastric pain, weight loss, dyspepsia, GI bleed- an ulcerating lesion; an infiltrating lesion (including ing, anemia, and evidence of gastric outlet obstruction. diffusely infiltrating or linitis plastica); and combined. (Figure 8.18A–D). Pathologic Features The architectural and cytologic morphologic hetero- geneity in gastric cancer has resulted in many different EGCs are usually relatively small (2–5 cm) and present histologic classifications and grading schemes, none of on the lesser curvature near the angularis. They are most which is universally accepted. The Lauren classification is often solitary. A macroscopic classification of early gas- most widely used by pathologists, and it classifies tumors tric neoplasia was developed in Japan in the 1960s (see histologically as intestinal, diffuse, or indeterminate/ Table 8.2); however, this classification scheme is not unclassified. The World Health Organization (WHO) widely used in Western countries. classification recognizes several other types and vari- Histologically, ECGs are usually well-differentiated, ants (see Table 8.1), in addition to those delineated in the and have glandular or papillary morphology that may be Lauren classification. Other classification schemes have difficult to distinguish from adjacent high-grade dysplasia also been proposed, including the Goseki system, which (Figure 8.17). Poorly differentiated and/or signet ring cell is based on gland formation and mucin production, and a morphology is unusual in this context. relatively new phenotypic classification scheme based on AGCs are typically solitary, and are located in the mucin immunohistochemistry. antrum or

antropylorus. Half measure 2 to 6 cm in great- Intestinal-type adenocarcinomas (Figure 8.19A–B), est dimension, and approximately 30% measure up to 10 as the name implies, are composed of neoplastic tubules cm. Only a minority of gastric carcinomas measure over and/or papillary structures with varying degrees of dif- ferentiation. Nuclear atypia is common, and mucin and necroinflammatory luminal debris are variably present. The tubular or glandular type is associated with older patient age, the formation of a mass lesion in the antrum, chronic H. pylori infection, gastric atrophy, and hematog- enous spread. Papillary carcinomas (Figure 8.19C–D) are also more common in older patients and are associated with hematogenous spread, especially to the liver, as well as with a high rate of lymph node metastases. Poorly cohesive carcinomas (including signet ring cell carcinoma, which should have at least 50% signet ring cells) are composed of infiltrating small nests or single cells (Figure 8.19E–F). This type is more often found in the gastric body and in younger patients; there is also an association with H. pylori infection, although the rela- tionship between H. pylori and poorly cohesive carcino- mas is not well understood. As in other sites in the GI tract, the cells have pale cytoplasm and an eccentrically placed nucleus. FIGURE 8.17 This early gastric carcinoma has a tubular Mucinous gastric adenocarcinomas should be at least pattern, and is confined to the mucosa. 50% mucinous. Similar to the colon, these carcinomas 8 Neoplasms of the Stomach 191 (A) (B) (C) (D) FIGURE 8.18 Advanced gastric adenocarcinomas have a wide variety of macroscopic appearances, including a polypoid or exophytic mass (A), an ulcerating lesion (B), an infiltrating lesion, including linitis plastica or diffuse infiltration of the gastric wall by tumor cells (C), or combined forms, such as this combined ulcerative and infiltrative tumor (D). consist of pools of mucin that contain floating strips or circumstance. Both the adenosquamous and squamous clusters of neoplastic epithelium, or single cells. subtypes have a very poor prognosis. Undifferentiated carcinomas lack morphologic and Gastric choriocarcinomas often feature a mixture cytologic differentiation entirely. This type most often of trophoblastic elements with other morphologic types enters into the differential diagnosis with lymphoma, mela- of gastric carcinoma. Tumors are typically very necrotic noma, or another type of tumor. and hemorrhagic (Figure 8.20A–B), and both immuno- There are several rare morphologic subtypes of gastric histochemical expression of β-HCG and elevated serum adenocarcinoma that comprise a small minority of cases. levels are characteristic. These very rare tumors have an Adenosquamous carcinoma of the stomach must have a extremely poor prognosis. neoplastic squamous component that comprises at least Hepatoid and alpha feto-protein (AFP)-producing 25% in addition to the glandular component. Pure squa- carcinomas are characterized by AFP production in the mous carcinomas of the stomach have been reported as context of either morphologic features that resemble hepa- well, but metastases must be rigorously excluded in this tocellular carcinoma (hepatoid), or a well-differentiated 19 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 8.19 This intestinal (tubular) type adenocarcinoma features well-differentiated tubules extending through the gastric wall (A). Another example shows greater nuclear atypia and luminal necroinflammatory debris (B). This papillary gastric adenocarcinoma forms papillary structures with prominent clear cell change, which is frequently seen in this type (C–D). Signet ring cell carcinomas should have at least 50% signet ring cells; the neoplastic cells have clear cytoplasm and an eccentrically placed nucleus (E). This poorly cohesive adenocarcinoma is composed of infiltrating cords of single cells that fill the lamina propria (F). tubulopapillary carcinoma with clear cell features and (EBV) infection, although the actual role of EBV in carcino- AFP production. The former closely resembles neoplastic genesis is controversial. Morphologically, these somewhat liver (Figure 8.20C–D), and variably expresses hepatocyte nodular tumors have a pushing border, and are composed antigen, AFP, and glypican-3. The morphology and immu- of sheets of cells with eosinophilic cytoplasm and numer- nophenotype can lead to diagnostic confusion, particu- ous tumor infiltrating lymphocytes (Figure 8.20E–F). larly when evaluating liver metastases or the possibility of These tumors are more common in the proximal stom- a primary liver tumor that has invaded the stomach. Both ach of Hispanic males, and although controversial, are morphologic types may be associated with high serum lev- believed to have a better prognosis. This subtype is also els of AFP, and both have an extremely poor prognosis. associated with previous subtotal gastrectomy. Medullary carcinoma (also known as lymphoepitheli- Other unusual subtypes include carcinosarcoma, pari- oma-like carcinoma or gastric carcinoma with lymphoid etal cell carcinoma, Paneth cell carcinoma, and malignant stroma) is strongly associated with Epstein–Barr virus rhabdoid tumor. 8 Neoplasms of the Stomach 19 3 (A) (B) (C) (D) (E) (F) FIGURE 8.20 This gastric choriocarcinoma is very necrotic and hemorrhagic, and the majority of the tumor is composed of choriocarcinoma (A–B). The patient had markedly elevated serum β-HCG, and the tumor stained with β-HCG as well. This hepatoid subtype of gastric adenocarcinoma closely resembles neoplastic liver (C–D). Medullary carcinomas feature nodules or clusters of tumor cells with numerous intratumoral lymphocytes (E–F). Peripheral lymphoid aggregates are commonly seen at the edges of the tumor. HEREDITARY DIFFUSE GASTRIC CANCER regardless of age. The presence of either of these features, AND OTHER GENETIC ASSOCIATIONS in addition to a diagnosis of diffuse gastric cancer at a young age (less than 40 years), or a family history of both Hereditary diffuse gastric cancer is an autosomal domi- lobular breast cancer and diffuse gastric cancer, should nant cancer syndrome characterized by signet ring cell car- prompt evaluation for germline CDH1 mutations, as they cinoma of the stomach, often in combination with lobular underlie a substantial proportion (30%–40%) of cases. breast carcinoma in women (see also Chapters 6 and 14). Patients with suspected hereditary diffuse gastric cancer Families with the syndrome are defined by the presence of require evaluation of peripheral blood DNA, usually in two or more documented cases of diffuse gastric cancer in the form of direct sequencing. first- or second-degree relatives, at least one of whom is Patients with hereditary diffuse gastric cancer usually diagnosed before age 50 years; or three or more cases of have normal or near-normal upper endoscopic evalua- diffuse gastric cancer in first- or second-degree relatives, tions, and often the microscopic lesions do not correlate 19 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide with a more visible or impressive lesion endoscopically. and 14. In brief, the molecular features of gastric adeno- Furthermore, gastroenterologists must biopsy widely, carcinoma are variable and related to tumor morphology. as microscopic foci of malignancy are typically present Intestinal-type carcinomas generally have molecular alter- within a macroscopically normal stomach. Lesions typi- ations similar to those of colorectal carcinomas, including cally consist of in situ foci of signet ring cell carcinoma, loss of heterozygosity or mutations affecting APC (30%– as well as surface signet ring cell change in some cases 40%), DCC (60%), KRAS (up to 30%), and TP53 (25%– (Figure 8.21A–B). Of note, both familial and sporadic 40%). In contrast, abnormalities affecting these genes are diffuse gastric cancers may show decreased, or absent, detected in less than 2% of diffuse-type carcinomas. Up to E-cadherin expression on immunohistochemistry, so this 20% of cases of gastric cancer show increased EGFR copy technique cannot be reliably used to identify patients with number by in situ hybridization, reflecting gene amplifica- germline mutations. The fact that there are no impressive tion or polysomy of chromosome 7. In addition, approxi- gross lesions may cause some clinicians to be cautious mately 25% of intestinal-type tumors overexpress HER2, about recommending prophylactic gastrectomy. However, compared to only 5% of diffuse-type carcinomas and 10% the lifetime risk for developing gastric adenocarcinoma is of tumors containing mixed intestinal and diffuse areas. extremely high (greater than 80% by age 80), and women Epigenetic alterations and promoter methylation are with CDH1 germline mutations also have an increased also more common among intestinal-type gastric adeno- risk of developing lobular breast cancer. Because of this carcinomas, and high-frequency microsatellite instability increased risk, as well as the difficulty in detecting early (MSI-H) is more frequently encountered in intestinal- lesions in these patients, prophylactic gastrectomy is usu- type carcinomas. Virtually all sporadic carcinomas with ally advised after age 20. MSI-H develop via hypermethylation of the MLH1 pro- Gastric adenocarcinoma is also associated with moter, similar to MSI-H colon cancers. However, gastric hereditary nonpolyposis cancer syndrome (more com- adenocarcinoma may also be a manifestation of Lynch monly in Asian patients); FAP (also more common in syndrome, and similar to their counterparts in the colon, Asian patients); Peutz–Jeghers syndrome; Li–Fraumeni morphologic features associated with gastric MSI-H syndrome, and gastric hyperplastic polyposis. tumors include tumor heterogeneity, abundant lymphoid stroma with intraepithelial lymphocytes (ie, medullary Ancillary Studies carcinoma), and mucinous differentiation. Although diffuse-type gastric adenocarcinomas The molecular features of gastric carcinoma and targeted have not been as extensively studied, more than 50% of molecular therapies are discussed in detail in Chapters 13 diffuse-type gastric adenocarcinomas show diminished (A) (B) FIGURE 8.21 Patients with hereditary diffuse gastric cancer typically have in situ foci of signet ring cell carcinoma, as seen here in the lamina propria (A, arrow; B). 8 Neoplasms of the Stomach 19 5 E-cadherin expression, reflecting abnormalities of CDH1 The most important independent prognostic indica- located on chromosome 16q22. tor for AGCs remains the anatomic stage (see the follow- ing section on grading and staging), as depth of invasion HER2 and lymph node status are critical predictors of prognosis. Some workers have argued that distally located carcino- Similar to adenocarcinomas of the esophagus and GE mas have a better prognosis than proximal tumors, but this junction, trastuzumab improves survival of patients with remains controversial. AGCs that overexpress HER2 when used in combination Gastric adenocarcinomas spread by three primary with conventional chemotherapy. Although data from the mechanisms: metastasis, direct extension, and peritoneal ToGA trial suggested that immunohistochemical overex- spread. Diffuse-type carcinomas are more likely to spread pression of HER2 correlates with response to trastuzumab throughout the peritoneum, whereas intestinal type tumors better than in situ hybridization, several studies since have are more likely to spread hematogenously. The prognosis shown comparable predictive values of these assays and for gastric adenocarcinoma in the West is poor, with a many laboratories now use either immunohistochemis- 1-year overall survival rate of 63%, and a 10-year survival try, in situ hybridization, or both to determine whether rate of only 10%. The survival rates are somewhat better patients with AGC should receive trastuzumab. in Japan, although earlier detection of lower-stage cancers There are two key differences between HER2 immu- may help to explain this difference. Female patients and nohistochemical testing in GE cancer and breast cancer. younger patients also have a better prognosis overall. The first is that there is more frequent HER2 staining AGCs are treated with complete surgical resection and heterogeneity in GE tumors. The second is that GE cases lymph node dissection; partial versus complete gastrectomy may demonstrate basolateral (U-shaped) or lateral mem- depends on the size and location of the tumor. A controver- brane staining rather than complete membrane staining, sial subject in the surgical management of gastric cancer is yet some of these cases are HER2-amplified by FISH. the recommended extent of lymph node dissection. Although Because of the heterogeneity, there are different scoring more extended lymph node dissections result in more accu- criteria for GE biopsies and resections, and any amount rate staging and possibly survival due to removal of diseased of 2+ or 3+ staining in a biopsy is considered equivo- nodes, there may be significantly increased morbidity and cal or positive, respectively, in esophagogastric cancers. mortality with extended dissections. Many patients also Because cases with basolateral and lateral membrane undergo adjuvant chemotherapy and radiation. staining may be HER2-amplified, the strict requirement for “complete membrane staining” that applies in breast cancer is relaxed. Grading and Staging There are many classification schemes for gastric carci- Management and Prognosis noma, none of which is universally used or accepted. From a practical perspective, many pathologists grade intesti- The two most important prognostic characteristics for nal-type gastric adenocarcinomas similarly to colorectal EGCs are overall size of tumor, and depth of invasion. adenocarcinomas, where well-differentiated tumors are Although larger tumors have an increased risk of submu- composed of well-formed glands or papillae

composed of cosal invasion, even very small tumors (less than 0.5 cm) more mature-appearing cells; moderately differentiated have invasive potential. Lymph node metastases occur tumors are composed of more complex architecture; and in 0% to 7% of intramucosal adenocarcinomas, but still poorly differentiated tumors have solid growth patterns, have an excellent (approximately 100%) 5-year survival very poorly formed glands, or single cells. By definition, rate. EGCs with submucosal invasion have an 8% to 25% signet ring cell carcinomas and diffuse-type gastric carci- rate of lymph node metastasis, and the 5-year survival rate nomas are considered poorly differentiated. is slightly lower but still quite high (80%–90%). Pathologic tumor staging of gastric carcinoma is per- EMR (or ESD) is the treatment of choice for EGCs, formed in accordance with the TNM staging manual of typically in concert with endoscopic ultrasound for opti- the American Joint Committee on Cancer (Table 8.3). The mal staging. Criteria for EGCs that are candidates for proximal 5 cm of stomach are included with and staged local excision include elevated lesions less than 2.0 cm; similarly to the esophagus (see Chapter 7). The proximal depressed lesions less than 1.0 cm that are not ulcerated; stomach is staged as detailed in Table 8.3, but well-differ- and documented absence of lymph node metastases. These entiated NETs, lymphomas, and sarcomas are not included specimens, similar to those from other sites in the GI tract, in this staging scheme. should be carefully evaluated grossly and entirely submit- Similar to the rest of the gastrointestinal tract, local ted with attention to margin status. When followed by extent of disease is assessed in the T category, whereas surveillance alone, over half of EGCs progress to advanced regional lymph node and distant metastases are classified carcinomas within 6 months to 7 years. in the N and M categories, respectively. 19 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 8.3 Pathologic Staging Criteria of Gastric degenerative/reactive in nature, and have been described Carcinomas in association with various types of gastritis and gastric ulcers. Distinguishing these entities is obviously critical, Primary Tumor (pT) because misdiagnosis of signet ring cell carcinoma may lead to significant and irreversible therapeutic interven- TX Primary tumor cannot be assessed T0 No evidence of primary tumor tions such as surgery and/or chemoradiation. Tis Carcinoma in situ: intraepithelial tumor without lamina Typically, signet ring cell change lacks nuclear atypia and propria invasion mitotic activity, although this is not always the case, particu- T1a Tumor invades lamina propria or muscularis mucosae larly in the context of highly reactive background mucosa T1b Tumor invades submucosa T2 Tumor invades muscularis propria (Figure 8.22A–B). Immunostains and special histochemical T3 Tumor invades subserosal connective tissues, without stains are somewhat helpful in the differential diagnosis, but involvement of visceral peritoneum or adjacent structures there are numerous pitfalls. Neoplastic signet ring cells are T4 Tumor invades visceral peritoneum or adjacent structures mucin positive, but the cells of signet ring cell change may T4a Tumor invades visceral peritoneum T4b Tumor invades adjacent structures be either positive or negative for mucin. Cytokeratins (CK) Regional Lymph Nodes (pN) NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastases N1 Metastasis in 1–2 regional lymph nodes N2 Metastasis in 3–6 regional lymph nodes N3 Metastasis in 7 or more regional lymph nodes N3a Metastasis in 7–15 regional lymph nodes N3b Metastasis in 16 or more regional lymph nodes Distant Metastases (pM) M0 No distant metastases M1 Distant metastases Source: Adapted from AJCC Cancer Staging Manual, 7th Edition, 2010. Because the depth of tumor invasion in early gastric (A) carcinomas predicts the likelihood of regional lymph node metastasis, it is helpful to report the depth of tumor inva- sion (as well as the T level) in EMR and ESD specimens. Differential Diagnosis and Diagnostic Challenges The diagnosis of most cases of gastric adenocarcinoma is relatively straightforward, particularly those with intestinal morphology. Adenocarcinomas of other organs can occa- sionally metastasize to the stomach (see subsequent sec- tion on metastases), and correlation with imaging studies, patient history, and immunohistochemical studies is often helpful. Poorly differentiated gastric adenocarcinomas may be much more problematic, and entities such as melanoma, lymphoma, and epithelioid mesenchymal tumors may enter into the differential diagnosis. Most of these cases can also (B) be resolved by a carefully selected panel of immunostains. A number of reactive conditions can also mimic FIGURE 8.22 Signet ring cell change, or pseudo- gastric adenocarcinoma. One of the most challenging is signet ring cells, are seen in a biopsy of a hyperplastic signet ring cell change a benign condition that morpho- gastric polyp with marked degenerative and inflammatory logically mimics signet ring cell carcinoma, particularly changes (A, B). Note that the dyscohesive atypical cells in biopsy specimens. These cells have also been referred are all sloughing from glands, and none are present in to as “pseudo-signet ring cells.” They are believed to be areas of intact lamina propria. 8 Neoplasms of the Stomach 197 will mark both neoplastic signet ring cells and signet ring malignancy in the context of a gastric ulcer with marked cell change, as both are epithelial. In contrast to signet inflammation and granulation tissue. Gastritis cystica ring cell carcinoma, the cells of signet ring cell change are profunda can occasionally mimic well-differentiated ade- often strongly positive for E-cadherin and negative for p53. nocarcinoma as well. Similar to colitis cystica profunda, However, reactive epithelia may be strongly positive for pro- the benign appearance of the epithelium, surrounding liferation markers, and E-cadherin stains may be difficult lamina propria, and noninfiltrative rounded contours of to interpret due to high background staining. If the atypical the misplaced epithelium help to confirm a diagnosis of cells are confined to glands on reticulin stains this may also gastritis cystica profunda/polyposa (Figure 8.23A–B). be helpful, as pseudo-signet ring cells typically are confined within glands and lack an infiltrating growth pattern into Metastases to the Stomach the lamina propria; however, reticulin may not be helpful if all the worrisome cells are detached, or if the worrisome cells Gastric metastases are relatively rare. However, when are in the context of an ulcer where normal glandular archi- present, they tend to be solitary and produce either a large tecture is severely disrupted. Occasionally, muciphages in the mural mass or ulcer, and thus may mimic a primary gas- lamina propria may also mimic signet ring cell carcinoma, tric malignancy. The most common tumors metastasiz- but the nuclei are typically small and uniform, and mac- ing to the stomach include melanoma (Figure 8.24A–B), rophage immunohistochemical markers make this a much breast carcinoma, and lung carcinoma, but metastases easier distinction. from virtually every site in the body have been reported in Several reactive conditions may also enter into the dif- the literature. Metastatic breast carcinoma can be particu- ferential diagnosis with gastric adenocarcinoma. Ulcers larly problematic, as it can present with a diffuse growth often contain epithelial regenerative changes that mimic pattern with signet ring cells that closely mimics gastric dysplasia, including nuclear enlargement and hyperchro- adenocarcinoma (Figure 8.25A–B). As breast carcinoma masia, prominent nucleoli, and increased mitoses, par- is positive for GATA3, GCDFP-15, ER, and PR, and nega- ticularly when there is marked acute inflammation. These tive for CDX2 and CK20, these markers can be helpful in inflammatory changes tend to be uniform and show making a diagnosis; however, it is important to remem- a gradual transition to more normal-appearing areas, ber that some gastric cancers are CK7+, and some do not whereas dysplasia is sharply demarcated from adjacent mark with CK20 either. The presence of an adjacent dys- non-neoplastic mucosa. In addition, exuberant granula- plastic lesion favors a primary gastric cancer, but these tion tissue with prominent endothelial cells can mimic may not be sampled or may be overgrown by the invasive a proliferation of malignant glands. For these reasons, component, so the absence of an adjacent dysplasia does caution should be used when entertaining a diagnosis of not exclude a p rimary gastric tumor. (A) (B) FIGURE 8.23 The entrapped submucosal epithelium seen in gastritis cystica profunda can occasionally mimic well- differentiated adenocarcinoma as well. Similar to colitis cystica profunda, the benign appearance of the epithelium, surrounding lamina propria, and noninfiltrative, rounded contours of the misplaced epithelium argue against a diagnosis of malignancy (A–B). Courtesy Dr. Andrew Bellizzi. 19 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 8.24 Metastatic malignant melanoma can closely mimic a poorly differentiated gastric adenocarcinoma (A–B). (A) (B) FIGURE 8.25 Metastatic breast carcinoma can closely mimic gastric adenocarcinoma. Tumor cells diffusely permeate the mucosa (A–B). The two can be indistinguishable histologically, thus ER and/or GATA3 immunostains may be very helpful. NEUROENDOCRINE NEOPLASMS (NECs). The term NEC encompasses small cell and large cell neuroendocrine carcinoma, as well as mixed small Neuroendocrine neoplasms are discussed in detail in and large cell carcinomas. This classification scheme also Chapter 3. This section will focus on aspects particular to contains the category “mixed adenoneuroendocrine car- gastric neuroendocrine neoplasia. cinoma” (MANEC), defined as a mixed tumor with at least 30% of each component (malignant epithelial and Terminology neuroendocrine). Well-differentiated NENs (formerly known as carci- noid tumors in the tubular gut) are referred to as neu- Epidemiology and Demographics roendocrine tumors (NETs) in the 2010 World Health Approximately 6% of GI NETs arise in the stomach; Organization Classification, and poorly differentiated the stomach is the second most common location within NENs are referred to as neuroendocrine carcinomas the GI tract, following jejunum/ileum and rectum. The 8 Neoplasms of the Stomach 19 9 incidence of NETs appears to be rising, as well, due at composed of ECL-cells, although approximately 25% are least in part to increased recognition and reporting. The composed of alternative cell types (eg, gastrin, somatosta- male to female ratio is essentially equal for gastric NETs tin, or serotonin-producing). Type III tumors are inher- (0.9:1), and the median age at presentation is 63 for both ently aggressive (less than 50% five-year survival), and men and women. Up to a quarter of extrapulmonary vis- tend to invade the gastric wall and demonstrate frequent ceral NECs arise in the GI tract, and NECs reportedly lymphovascular invasion. They are managed with resec- account for 6% to 16% of gastric neuroendocrine neo- tion, usually gastrectomy, and regional lymph node dis- plasms. NECs are more common in men, but the age of section. Occasionally, very small polypoid type III tumors presentation is similar to that seen in gastric NET. can be followed with polypectomy and extremely rigorous clinical surveillance. Clinical Features Gastric NETs arise in three distinctive clinical settings, Pathologic Features with implications for prognosis and management. Type I tumors, representing 70% to 80%, arise in the setting In the stomach, intramucosal tumors greater than 500 of autoimmune atrophic gastritis (Figure 8.26). Type μm and submucosally invasive tumors are designated as II tumors (5%–10%) arise in association with MEN1– NETs, while lesions measuring 150 to 500 μm are termed Zollinger–Ellison syndrome. Type III tumors (20%–25%) neuroendocrine dysplasias, and those less than 150 μm are arise sporadically. Overall, the majority of gastric NET classified as hyperplasias. The distinction of these seems (76%) have localized disease at the time of presentation. arbitrary, as lesions as small as 200 μm have been shown Type I and II tumors are found in the gastric body, to have neoplastic potential based on molecular studies. typically confined to the mucosa or submucosa, and have Grossly, NETs are usually well-circumscribed, with a tendency to be multifocal. They are composed of ECL- a fleshy and homogeneous cut surface. The majority of cells (ie, histamine-producing) and are driven by hyper- the tumor is often in the submucosa and muscular wall, gastrinemia. Hypergastrinemia-driven tumors, especially although polypoid lesions may be covered by mucosa and type I, are typically indolent (100% and 60%–90% five- protrude into the lumen. Mucosal ulceration and necrosis year survival, respectively, for types I and II). Patients may be macroscopic signs of aggressive behavior. with small type I and II tumors may be managed with Histologically, NETs are characterized by a variety endoscopic removal and surveillance, while patients with of organoid growth patterns (discussed and illustrated in type II tumors additionally benefit from removal of the detail in Chapter 3), including nested, trabecular, gyri- gastrinoma, in which case their NET may spontaneously form, glandular,

tubuloacinar, pseudorosette-forming, regress. solid, and mixed. The nuclear chromatin is typically Type III tumors are nearly always solitary and may granular and/or speckled, often referred to as “salt and arise anywhere in the stomach. They, too, are generally pepper.” Nucleoli are generally inconspicuous. Cells are typically monomorphic with moderate to abundant cyto- plasm. The neuroendocrine nature of these neoplasms is usually evident on hematoxylin and eosin (H&E) staining, but can be confirmed with immunohistochemical staining for neuroendocrine markers. NECs have morphologic features similar to those seen in other sites (see Chapter 3). Mitotic activity and necrosis are usually conspicuous, as is Azzopardi effect. Many gastric NECs have an admixed component of ade- nocarcinoma along with the neuroendocrine component, and adjacent epithelial dysplasia and/or intramucosal carcinoma is often present as well. As with NETs, the diagnosis can be supported with general neuroendocrine markers. Most MANECs consist of NEC with a coexistent component of adenocarcinoma (Figure 8.27A–E). The WHO specifically states that adenocarcinomas in which scattered neuroendocrine cells are identified immunohis- tochemically are not considered to be MANECs. In addi- FIGURE 8.26 The majority of gastric NETs arise in tion, the non-neuroendocrine component must be overtly the context of autoimmune atrophic gastritis; note the cytologically malignant, and care must be taken not to adjacent intestinal metaplasia and inflammation. confuse entrapped glands with a glandular component. 2 0 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 8.27 Mixed adenoneuroendocrine carcinomas (MANEC) are defined as a mixed tumor with at least 30% of adenocarcinoma and NEC (A). The adenocarcinoma component (B) is negative for chromogranin (C). Morphologically distinct areas of neuroendocrine differentiation (D) show strong, diffuse chromogranin positivity (E). Courtesy Dr. Andrew Bellizzi. 8 Neoplasms of the Stomach 2 01 Grading and Staging Grading of neuroendocrine neoplasms is discussed in detail in Chapter 3. Staging of gastric NET is summarized in Table 8.4. Staging of gastric NEC and MANEC is per- formed similarly to gastric adenocarcinomas (see previous section grading and staging on page 195). Differential Diagnosis The primary entities in the differential diagnosis of NET include non-NECs and (rarely) lymphoma; fortunately, the distinction can be made easily by immunohistochemistry in most cases. NETs with glandular, acinar, or pseudoro- sette patterns are most likely to mimic adenocarcinoma, whereas tumors with nested growth may be mistaken for squamous cell carcinoma or solid-pattern adenocarci- noma. It is also important to note that NETs may produce FIGURE 8.28 NETs can produce both stromal and luminal or stromal mucin (Figure 8.28), a further mimic luminal mucin, which can lead to confusion with gastric of adenocarcinoma. NETs are characterized by relative adenocarcinoma. monomorphism and the characteristic nuclear chromatin pattern, while non-NECs tend to exhibit greater pleomor- phism. When the diagnosis is in question, pathologists should consider NET and have a low threshold for order- Tumor cells lack the classic “speckled” pattern of neu- ing IHC for general neuroendocrine markers. roendocrine chromatin as well. Demonstration of strong Gastric glomus tumors (see Chapter 5) may also be smooth muscle actin (SMA) expression is useful in secur- mistaken for neuroendocrine tumors, especially since ing the diagnosis of glomus tumor. they often express synaptophysin (albeit weakly). Glomus The primary entities in the differential diagnosis of tumors are often multinodular and/or plexiform, and NEC also include non-NECs, as well as other high-grade tend to grow within blood vessel walls (subendothelially). round blue cell tumors (including lymphoma, melanoma, and some sarcomas), and occasionally chronic inflam- mation in crushed specimens. NECs should feature char- acteristic cellular morphology and demonstrate diffuse TABLE 8.4 Staging of Well-Differentiated Gastric expression of at least one general neuroendocrine marker Neuroendocrine Tumors (NETs) in addition to broad-spectrum keratins, which may appear Primary Tumor (T) perinuclear or dot-like. Significant pleomorphism is more suggestive of non-NEC. CD45 is helpful in excluding lym- TX Primary tumor cannot be assessed phoma, and S100 for excluding melanoma. In crushed T0 No evidence of primary tumor small biopsies, IHC for broad-spectrum keratins (and/or Tis Tumor less than 0.5 cm, confined to mucosa T1 Tumor invades lamina propria or submucosa general neuroendocrine markers) and CD45 may be used and 1.0 cm or less in size to distinguish NEC from chronic inflammation. T2 Tumor invades muscularis propria or more than 1.0 cm in size T3 Tumor penetrates subserosa T4 Tumor invades visceral peritoneum (serosa) or MESENCHYMAL NEOPLASMS other organs or adjacent structures Mesenchymal neoplasms, including the differential diag- Regional Lymph Nodes (N) noses and immunohistochemical and molecular features, are discussed in detail in Chapter 5. This section will focus NX Nodes cannot be assessed N0 No regional lymph node metastases on those mesenchymal tumors that are most common to N1 Regional lymph node metastases the stomach. Distant Metastases (M) Gastrointestinal Stromal Tumors M0 No distant metastases M1 Distant metastases Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the GI tract, and the Source: Adapted from AJCC Cancer Staging Manual, 7th Edition, 2010. majority of them are located in the stomach (60%–70% of 2 0 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide tumors). GISTs are believed to arise from the KIT-positive with a worse prognosis. Gastric GISTs located in the fun- interstitial cells of Cajal, which are an innervated network dus and cardia have a worse prognosis than those located of cells associated with Auerbach’s plexus that coordinate in the antrum. peristalsis. Although symptoms may include nonspecific Morphologically, gastric GISTs have a spindle cell pat- upper abdominal pain, GI bleeding, bloating, early satiety, tern approximately 50% of the time, an epithelioid phe- and signs/symptoms of a mass, a significant proportion notype approximately 40% of the time, and are mixed are incidental findings on imaging studies or procedures pattern 10% of the time. Nuclear palisading and cytoplas- for unrelated conditions. In the stomach, GISTs are more mic vacuolization are also more commonly seen in gastric likely to occur in older patients (median age 63). GISTs (Figure 8.29A–B). A number of important genetic syndromes involve SDH-deficient GISTs (also known as pediatric GIST gastric GISTs. Carney triad includes epithelioid gas- or type 2 GIST) typically occur in female children and tric GIST, pulmonary chondroma, and paragangliomas; adolescents with a peak age of onset in the second decade. GISTs in this context are wild-type, and believed to be Most tumors develop in the stomach or omentum. This part of the spectrum of succinate dehydrogenase (SDH) subtype accounts for approximately 5% to 7% of gastric deficient GISTs. Patients with Carney–Stratakis syndrome GISTs. The distinctive histology features a multinodu- have epithelioid gastric GISTs and paragangliomas; these lar or plexiform growth pattern (Figure 8.30A), often patients also have germline SDH subunit gene mutations with epithelioid morphology. These tumors are positive and thus are part of the spectrum of SDH-deficient GISTs for KIT, but virtually all demonstrate a loss of SDHB as well. staining by immunohistochemistry, and thus this stain is a good screening tool for this tumor. Only a minor- Pathologic Features ity of these tumors have an identifiable SDH mutation, however. Typical grading criteria are not applicable to Grossly, GISTs arise in the muscularis propria, and are this subtype (see the following section on grading and usually somewhat centered in the wall of the stomach. risk stratification), and they are often imatinib resistant. They vary widely in size, ranging from 1.0 mm to more Additionally, this subtype is more likely to metastasize than 40 cm, with a median size of 6 cm in the stom- to lymph nodes, although this finding does not appear to ach. Tumors may protrude into the lumen in a polypoid affect prognosis. fashion, with overlying mucosal ulceration. The cut sur- There is also a subset of gastric GISTs with PDGFRA face may show hemorrhage, calcification, or, less com- mutations (particularly exon 18) that have an extremely monly, necrosis. GISTs are typically circumscribed, and indolent course, even when large. These tumors typically although the overlying mucosa is often ulcerated, true are purely epithelioid or have an epithelioid component, invasion of the mucosa is uncommon, and is associated and have low or no mitotic activity. (A) (B) FIGURE 8.29 Gastric GISTs are more likely to be epithelioid, and perinuclear vacuoles are frequent (A). Nuclear palisading can be striking in gastric GISTs (B). 8 Neoplasms of the Stomach 2 0 3 (A) (B) FIGURE 8.30 SDH-deficient GISTs have a distinctive multinodular or plexiform growth pattern (A), typically with epithelioid morphology (B). Ancillary Studies harbor BRAF V600E mutations; these are KIT positive by immunohistochemistry. A detailed discussion of the immunohistochemical features of GISTs is presented elsewhere (Chapters 5, 13, and 14). In brief, 95% of GISTs are KIT-positive. Approximately Grading, Risk Stratifi cation, and Prognosis 5% of GISTs are KIT-negative; the majority of these are Grading, risk stratification, and prognosis are discussed epithelioid GISTs that have PDGFRA mutations, and in detail in Chapter 5. Of note, the risk of aggressive these typically arise in the stomach (as noted in the pre- behavior in gastric GISTs is relatively low until tumors ceding section on pathologic features) or are extraintes- reach more than 10 cm in size. As mentioned previously, tinal. Most GISTs (~99%) also mark with DOG1 (also SDH-deficient GISTs have a tendency to metastasize to known as ANO-1). The majority of KIT-negative GISTs lymph nodes (approximately half of cases), yet overall stain with DOG1 as well, including 79% of those with their behavior is indolent even when lymph node metas- PDGFRA mutations (as opposed to KIT which only stains tases occur. Because of this unusual behavior, standard 9% of GISTs that have a PDGFRA mutation). Caveats criteria for grading and risk stratification are probably not regarding the use of KIT and DOG1 are discussed in detail applicable to this subgroup of tumors, and it has been rec- in Chapter 5. ommended that conventional criteria not be used in this Approximately 80% of GISTs have KIT mutations, specific context. Detailed discussions of therapy for GISTs and up to 20% contain wild-type KIT. Nearly 10% of are presented in Chapters 5, 13, and 14. the latter harbor mutations in PDGFRA (platelet-derived growth factor A). This subset of GISTs is more likely to Differential Diagnosis occur in the stomach (as noted in the preceding section on pathologic features) or proximal small bowel, or to be A detailed discussion of the differential diagnosis of GIST extraintestinal. They are typically epithelioid and have an is presented in Chapter 5. In the stomach, the most com- indolent clinical course. Immunohistochemistry for KIT mon entity in the differential diagnosis is gastric schwan- in these tumors shows faint positive or negative staining. noma. Less common mesenchymal tumors that may enter Because certain mutations (both KIT and PDGFRA) may into the differential diagnosis at this site include inflam- affect prognosis, molecular analysis of GISTs is increas- matory fibroid polyp (IFP), glomus tumor, and plexiform ingly regarded as the standard of care. angiomyxoid tumor. Leiomyomas, granular cell tumors, Some wild-type GISTs lack both KIT and PDGFRA and benign neural polyps are rarely seen in the stom- mutations; these include the SDH-deficient GIST ach, as are neurofibromas; the latter are typically seen in described in the preceding section on pathologic features, association with NF1. Desmoid tumors, PEComas, and as well as those occurring in the setting of NF1, Carney inflammatory myofibroblastic tumors, which are also rare triad, and Carney–Stratakis syndrome. Some of these in the stomach, are discussed in detail in Chapter 5. 2 0 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Schwannoma vessels are common, and the tumor cells show a subendo- thelial growth pattern within walls of blood vessels. The The stomach is the most common site for GI schwan- cells are uniform and rounded, with clear to eosinophilic nomas, and these lesions are more common in women. cytoplasm and sharply defined cell borders. The nucleus Grossly, schwannomas are circumscribed but unencapsu- appears “punched out” and round, and is well demarcated lated tumors with a firm, rubbery, yellow–gray to tan cut from the surrounding cytoplasm. There is typically diffuse surface. Cystic change and hemorrhage are not usually reactivity for SMA, but KIT and DOG1 are negative, in seen. Schwannomas are moderately cellular tumors; focal contrast to GIST. Calponin and caldesmon are typically nuclear atypia is common, but mitotic figures should be positive as well, but desmin is often negative. Some tumors rare to absent, and

atypical mitoses should not be seen. are focally positive with CD34 and synaptophysin, but Unlike soft tissue schwannomas, palisading is rare in the chromogranin and keratin should be negative. GI tract, as are foamy histiocytes. A notable feature in GI tract schwannomas (as opposed to those that occur in the soft tissues) is a dense lymphoplasmacytic cuff, Infl ammatory Fibroid Polyp often with germinal centers, present at the periphery of the tumor (Figure 8.31A–B). Numerous lymphocytes may Inflammatory fibroid polyps (IFPs) are benign mesenchy- also be seen admixed with tumor cells. Epithelioid foci mal tumors that are commonly found in the antrum of are very rare in GI tract schwannomas. GI tract schwan- the stomach or the ileum, and often present as a polyp. nomas express S100 and generally lack immunoreactivity The neoplastic nature of these lesions has been debated, for KIT, DOG1, SMA, desmin, and other smooth muscle but some IFPs have been found to have activating muta- markers; however, rare tumors can show focal expres- tions in PDGFRA, so they in fact may be neoplastic. They sion of keratin, SMA, or desmin. In addition, unlike their present in a wide age range but are typically seen in adults soft tissue counterparts, GI tract schwannomas do not from 60 to 80 years old. These lesions arise from the express calretinin. submucosa, and the overlying mucosa is often ulcerated (Figure 8.32A–B). Tumors range in size from less than 0.5 cm to over 4.0 cm, with an average size of 1 to 2 cm. Glomus Tumor Histologically, they have ill-defined margins, and consist On the rare occasion when glomus tumors arise in the of a bland proliferation of spindle cells and stellate cells GI tract, they are most commonly seen in the antrum of with prominent admixed inflammatory cells, particularly the stomach. They typically arise in the muscularis pro- eosinophils. Blood vessels are usually prominent, and may pria, and feature cellular nodules or nests separated by show surrounding concentric “onion-skin” fibrosis. IFPs bands of smooth muscle extending from the muscularis are positive for CD34 and variably positive for SMA; they propria (see Chapter 5). Prominent slit-like and dilated are negative for KIT, desmin, and S100. (A) (B) FIGURE 8.31 Gastric schwannomas feature a dense lymphoplasmacytic cuff present at the periphery of the tumor (A). Focal nuclear atypia is common, and numerous lymphocytes may also be seen admixed with tumor cells (B). 8 Neoplasms of the Stomach 2 0 5 (A) (B) FIGURE 8.32 Inflammatory fibroid polyps arise from the submucosa (A), and typically have ill-defined margins. They consist of a bland proliferation of spindle cells with admixed inflammatory cells, particularly eosinophils, as well as prominent blood vessels that may show a surrounding cuff of plump lesional cells (B). Plexiform Fibromyxoma (Plexiform are negative for KIT, DOG1, and S100. Plexiform fibro- Angiomyxoid Myofi broblastic myxomas are rare, and thus knowledge of natural history Tumor of Stomach) is limited, but they appear to behave in a benign fashion. These tumors present as a mural mass in young to middle aged adults, are nearly exclusive to the gastric antrum, and are often mistaken for GISTs. They are bland, multi- HEMATOLYMPHOID NEOPLASMS lobular spindle cell tumors that are sharply circumscribed, have a plexiform growth pattern, and contain abundant The stomach is the most common primary site for GI myxoid stroma with prominent small blood vessels (Figure lymphomas to develop, and it may be involved by many 8.33A–B). Vascular invasion is common. These tumors of the lymphomas discussed throughout this text. In are positive for SMA and stain variably with desmin; they the stomach, as in the remainder of the GI tract, B cell (A) (B) FIGURE 8.33 Plexiform fibromyxoid tumors are bland, multiolobular spindle cell tumors (A) that are sharply circumscribed, have a plexiform growth pattern, and contain abundant myxoid stroma with prominent small blood vessels (B). 2 0 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide lymphomas far outnumber their T cell counterparts. The two most important gastric hematolymphoid neoplasms are mucosa- associated lymphoid tissue (MALT) lym- phoma and diffuse large B cell lymphoma (DLBCL); these will be discussed here in addition to important (if rela- tively rare) variants of these lymphomas. MALT Lymphoma This low-grade B cell lymphoma is more properly referred to as “extranodal marginal zone lymphoma of mucosa- associated lymphoid tissue” and was described by Isaacson and Wright in 1983. While MALT lymphoma can occur throughout the GI tract, the stomach is, by far, the most common site of involvement, with around 85% of all GI MALT lymphomas occurring here. In the stomach, most MALT lymphomas are intimately related FIGURE 8.35 Arising most often in a background to underlying infection with H. pylori, and arise from of H. pylori gastritis, MALT lymphoma frequently has so-called “acquired” mucosa-associated lymphoid tissue, admixed germinal centers, which can have a “moth-eaten” as discussed in Chapter 4. As such, the majority of cases appearance when colonized by lymphoma cells. Note the can actually be treated by eradication of the underlying prominent, expanded marginal zone composed of pale- infection. staining cells around the germinal centers (arrows). Pathologic Findings The cells comprising MALT lymphomas are small, and and individually scattered. One may also see reactive ger- may resemble either centrocytes of the follicle center, or minal centers (similar to those seen in H. pylori gastri- have more abundant, pale cytoplasm and indented nuclei, tis), which are infiltrated or colonized by the lymphoma giving them a “monocytoid” appearance (Figure 8.34). cells, sometimes creating a “moth-eaten” or “naked” Most MALT lymphomas also contain some large cells appearance (Figure 8.35). In addition, the lymphoma cells resembling centroblasts, although these are in the minority commonly infiltrate and disrupt normal mucosal struc- tures (glands and pits), creating lymphoepithelial lesions (Figure 8.36A–B). These are a characteristic feature of MALT lymphomas, although they are nonspecific, and similar destructive infiltration may be seen in other types of lymphoma. In addition, infiltration of the epithelium by benign lymphocytes can be seen in a variety of reactive conditions. While scattered large, centroblast-like cells are common in MALT lymphoma, large collections (greater than 20 or so together) or sheets of such cells should raise the diagnosis of DLBCL, which will be discussed in a subsequent section. Some cases of MALT lymphoma have plasmacytic differentiation, in which clonal plasma cells comprise a component of the infiltrate. Occasionally, there may be essentially complete plasmacytic differentia- tion, an appearance that can lead to diagnostic difficulty (Figure 8.37). Ancillary Studies FIGURE 8.34 MALT lymphoma is usually composed Immunohistochemically, MALT lymphoma expresses of predominantly small lymphocytes, many of which pan-B cell markers such as CD20 and CD79a (Figure 8.38). have ample cytoplasm and indented nuclei (arrows), Between 30% and 50% of MALT lymphomas are pur- an appearance that has been termed “monocytoid.” A ported to aberrantly coexpress CD43 on the malignant number of gastric glands (arrowhead) are destroyed by B cells, though this is not specific, as it may be seen in infiltrating lymphoma cells. other types of lymphoma including mantle cell lymphoma 8 Neoplasms of the Stomach 2 07 FIGURE 8.36 Though not specific to MALT lymphoma, FIGURE 8.38 This CD20 immunostain highlights the lymphoepithelial lesions (LELs) are a hallmark, created diffuse infiltrate of neoplastic B cells in a gastric MALT when lymphoma cells invade and destroy the epithelial lymphoma. structures of mucosa, in this case, gastric glands (A, arrow). The use of a cytokeratin immunostain (B) highlights the destruction, revealing scattered remnant epithelial cells among the infiltrate (arrow). Courtesy to the process, it will likely express either monotypic Dr. Henry Appelman. kappa or lambda light chain, which can be a helpful indi- cator of clonality when it is present (Figure 8.39). While there is no specific immunohistochemical phe- (MCL) and chronic lymphocytic leukemia/small lympho- notype that points to MALT lymphoma, there are a num- cytic lymphoma (CLL/SLL). In fact, there is no specific ber of associated molecular abnormalities that can be immunohistochemical marker for MALT lymphoma, seen. The t(11;18)(q21;q21) translocation is the most com- meaning that the diagnosis relies fairly heavily on the mor- mon translocation found in GI MALT lymphomas, bring- phologic impression. If there is a plasmacytic component ing together the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18. This translocation may FIGURE 8.37 Some MALT lymphomas, in this case an example in the colon, have extensive or even complete plasmacytic differentiation. When the cells have the FIGURE 8.39 This colonic MALT lymphoma had appearance of mature plasma cells like this, it can lead to extensive plasmacytic differentiation, including many cells diagnostic difficulty, being easily confused with a chronic with crystallized immunoglobulin in their cytoplasm (“Mott inflammatory condition or a plasma cell neoplasm such as cells”). This kappa light chain immunostain confirms their plasma cell myeloma. monotypic/clonal nature. 2 0 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide be seen in cases arising in the setting of H. pylori infection lymphoma. Interestingly, there are reports of MALT lym- or in cases that are H. pylori negative, but in either case, phomas outside the stomach, including in the small intes- its presence is associated with resistance of the lymphoma tine and even in the colon, responding to similar therapy. to conservative therapy aimed at eradication of the infec- Importantly, while standard follow-up biopsies to assess tion. Other molecular abnormalities reported in MALT eradication of H. pylori typically occur in the 4 to 6 week lymphoma include t(14;18)(q32;q21) [IgH/MALT1], timeframe, the lymphoid infiltrate is likely to be unchanged t(1;14)(p22;q32) [BCL10/IgH], and trisomy 3q27. There at this point in time, even in patients who successfully is debate about whether to test for the t(11;18) a priori undergo treatment and achieve remission. In fact, there when making a diagnosis of MALT lymphoma in order are reports of complete resolution of the atypical lymphoid to predict the small subset of patients unlikely to respond population taking many months, even more than a year. to conservative therapy. There is currently no clear recom- Thus, it is important to be sure that treating physicians are mendation for or against this practice. aware of this fact to avoid chemoradiation or other aggres- sive therapies for a lymphoma that would likely respond Differential Diagnosis and to conservative therapy if given enough time. Comparison Diagnostic Challenges of follow-up biopsies to the pretreatment tissue and/or to earlier follow-up samples can be very helpful, with a diag- The differential diagnosis for MALT lymphoma includes a nosis such as “residual MALT lymphoma, improved from number of small B cell neoplasms such as MCL (discussed [DATE]” providing useful information to the clinician. in detail in Chapter 11) and follicular lymphoma (discussed While it does not affect the stomach, there is a variant in Chapter 9). Both can have a nodular pattern that can of MALT lymphoma affecting the small intestine that mer- overlap with the appearance of the reactive germinal centers its mention. Immunoproliferative small intestinal disease that can be seen in MALT lymphomas, though immunohis- (IPSID) is a lymphoma with complete or nearly complete tochemical stains are usually sufficient to make the distinc- plasmacytic differentiation that preferentially involves the tion, with the cyclin-D1 expression of MCL and the BCL2 small intestine and causes malabsorption and weight loss positivity of the neoplastic follicles in follicular lymphoma (Figure 8.40). The plasma cells are CD138 positive, and are being most helpful. Plasmacytic differentiation in MALT lymphoma can lead to confusion with true plasma cell neo- plasms, and this can be difficult to sort out when there is complete plasma cell differentiation. Correlation with other systemic manifestations of disease, such as a search for a monoclonal serum protein, may help to identify a plasma cell neoplasm like myeloma, but it may be necessary simply to provide a differential diagnosis in such situations. Perhaps the most challenging situation is discerning when an intense H. pylori gastritis has “crossed the line” and become a lymphoma. Aberrant coexpression of CD43 by the B lymphocytes is useful but, unfortunately, there is no specific immunohistochemical marker for lymphoma; it thus is often a matter of morphologic evaluation and judgment. Molecular studies for B cell gene rearrange- ments may also be helpful, but the presence of clonality does not unequivocally imply a diagnosis of malignancy. In addition to the histologic evidence provided by a monot- onous and destructive infiltrate

of CD20-positive B cells, it is important to take into account the clinical impression. For example, a malignant-appearing ulcer or macroscopic evidence of an infiltrative process such as thickened muco- sal folds may prove helpful in making the diagnosis. In addition, such findings give the endoscopist a “target” to FIGURE 8.40 Immunoproliferative small intestinal disease assess on follow-up examinations after therapy. (IPSID) is considered a variant of MALT lymphoma that may The association with H. pylori infection makes MALT be associated with underlying Campylobacter infection. lymphoma amenable to conservative therapy aimed at erad- IPSID is composed almost entirely of plasma cells, at least ication of the inciting infection. The vast majority of gastric in early stages of the process. Patients may also have a MALT lymphomas will respond to this therapy, though monoclonal serum protein, composed of a truncated IgA heavy chain and leading to the alternate name for the those harboring the t(11;18) have a high prevalence of process, “IgA heavy chain disease.” resistance, as described in the preceding section on MALT 8 Neoplasms of the Stomach 2 0 9 often immunohistochemically positive for immunoglobu- rate and immunohistochemical staining pattern should lin A (IgA) heavy chain. In about half of the cases, there is still be present. When judging cell size, endothelial cells an associated production of an abnormal, truncated IgA in the vessels in and around a DLBCL are a convenient heavy chain that can be found in the serum, leading to the metric, as their nuclei are a good measure of “large” size alternative name “IgA heavy chain disease.” IPSID is most (Figure 8.42). By definition, the cells of DLBCL should common in young men and has a strong geographic asso- be at least the size of a macrophage nucleus and at least ciation, occurring in the Middle East, the Mediterranean twice the size of a normal lymphocyte nucleus. DLBCL is region, and the Cape region of South Africa. It tends to be a destructive neoplasm, in which the malignant cells infil- associated with low socioeconomic status and may result trate widely through the tissue, obliterating normal struc- from a chronic infection, perhaps with a Campylobacter tures and, in the GI tract, often leading to a mucosal ulcer species; early cases have been shown to respond to broad- (Figure 8.43A–B). A large number of cases present at high spectrum antibiotics. Many cases, however, present at stage, reflecting the aggressiveness of the process. In addi- advanced stage and IPSID may progress to a high-grade tion to the most common morphology described earlier lymphoma indistinguishable from DLBCL. in this paragraph (technically, the “centroblastic variant” due to the resemblance of the cells to normal centroblasts Diffuse Large B Cell Lymphoma of the germinal center), there are two other fairly common morphologic variants. The first is the immunoblastic vari- DLBCL is the most common lymphoma affecting the GI ant, in which the cells have a more uniform appearance tract, and the stomach is a common site of involvement. with one large, centrally located nucleolus (Figure 8.44). It may arise de novo, or evolve from a precursor low- DLBCL may also have an “anaplastic” appearance, with grade lymphoma, such as follicular lymphoma or MALT cells that are much more pleomorphic (Figure 8.45), lymphoma. though it is important to understand that this variant is unrelated to anaplastic large cell lymphoma, which is a Pathologic Features T cell process. In elderly patients, a subtype of DLBCL can be associated with EBV in a manner analogous to DLBCL is characterized morphologically by diffuse clus- EBV-driven lymphoproliferative disorders in immunosup- ters (usually defined as more than 20 cells together) and/ pressed patients who have undergone organ transplants or sheets of large, dyscohesive cells with vesicular nuclei (so-called monomorphic post-transplant lymphoprolifera- and one or more inconspicuous nucleoli (Figure 8.41). tive disorders). In both of these cases, the lymphoma is Occasionally, the cells of DLBCL will be more “interme- essentially indistinguishable from conventional DLBCL, diate” in size, which can cause some diagnostic confusion, though there can be relatively more immunoblast-like cells though the other features such as a rapid proliferative and even large atypical cells resembling Reed–Sternberg FIGURE 8.42 As an internal gauge of “large” cell size, FIGURE 8.41 Diffuse large B cell lymphoma (DLBCL) is endothelial cells from vessels admixed with a lymphoma most frequently composed of large cells resembling the provide a convenient comparison. Note the similarity centroblasts of the normal germinal center, with vesicular of this endothelial nucleus (arrow) to the nuclei of the nuclei and fairly inconspicuous nucleoli. surrounding lymphoma cells. 210 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 8.45 The anaplastic variant of DLBCL has very FIGURE 8.43 DLBCL is a destructive process, pleomorphic cells, many of which show nuclear lobes obliterating normal structures of the wall of a viscus (A) and/or indentation (arrows). While the morphology can and often leading to mucosal ulcer (B) as evidenced by be similar, the anaplastic variant of DLBCL is completely overlying ulcer exudate (arrow). unrelated to anaplastic large cell lymphoma, which is a T cell process. cells in Hodgkin lymphoma (Figure 8.46). Evidence of EBV involvement such as a positive EBV-encoded ribonu- cleic acid (EBER; in situ hybridization) probe is useful in DLBCL may have a germinal center cell phenotype, with the diagnosis (Figure 8.46, inset). expression of CD10 and/or BCL6, or a so-called “acti- vated B cell” phenotype, with expression of MUM-1 and other markers. Perhaps 10% of cases aberrantly coexpress Ancillary Studies CD5, a T cell marker, which can lead to confusion with Immunohistochemically, most DLBCLs express pan-B the blastoid or pleomorphic variants of MCL, discussed in cell markers including CD20, PAX-5, and CD79a detail in Chapter 11. Unlike MCL, DLBCL is negative for (Figure 8.47). BLC-2 is commonly expressed as well, and cyclin-D1 expression. FIGURE 8.44 The immunoblastic variant of DLBCL FIGURE 8.46 Scattered Reed–Sternberg-like cells consists of somewhat uniform cells that have prominent, (arrows) are present in an EBV-driven lymphoma in an central nucleoli (arrow), reminiscent of normal elderly patient. A positive EBER probe (in situ hybridization immunoblasts. for EBV) confirms the diagnosis (inset). 8 Neoplasms of the Stomach 211 FIGURE 8.47 Immunostains for CD20 are diffusely FIGURE 8.48 Strong nuclear staining for Ki-67 reveals a positive in a DLBCL. very high proliferative rate in this “double-hit” lymphoma. From a molecular standpoint, DLBCL may harbor MALT lymphoma, and distinction of early DLBCL can be a number of abnormalities, often reflecting the presence diagnostically difficult. While a large collection or sheet(s) of of an underlying low-grade process. As such, the t(14;18) large cells in the setting of something recognizable as MALT translocation characteristic of follicular lymphoma is lymphoma was termed “high-grade MALT lymphoma” present in up to one-third of cases. Other abnormalities in the past, this designation is now out of date, and such include BCL6 gene alterations (chromosome 3q27) and, an appearance should currently be diagnosed as DLBCL. for those with an activated B cell phenotype, molecular Other entities in the differential diagnosis include blastoid aberrancies along the NF-κB pathway, leading to consti- or pleomorphic MCL as described in the preceding section tutive activation and inhibition of apoptosis. As discussed on ancillary studies, Burkitt lymphoma (usually BCL2 neg- in Chapters 4 and 9, a group of neoplasms with features ative and harboring MYC abnormalities), and B lympho- intermediate between DLBCL and Burkitt lymphoma, blastic leukemia/lymphoma, which expresses markers of the so-called “double-hit” lymphomas, have a germinal immaturity such as CD34 and terminal deoxynucleotidyl center immunophenotype with CD10 expression. They harbor abnormalities in MYC, along with BCL2 and/or BCL6. Such cases usually have a high proliferative rate (Figure 8.48) and their distinction seems to be important from a prognostic, and possibly therapeutic, standpoint in younger patients, as there is a subset of cases with MYC and BCL2 abnormalities that are treatment-refractory. Differential Diagnosis and Diagnostic Challenges The differential diagnosis of DLBCL includes a number of other B cell lymphoproliferative disorders. Perhaps most important is the differentiation from (or recognition of superimposition upon) an underlying low-grade lymphoma. This is crucial because de novo DLBCL, as an aggressive lym- phoma, is potentially curable by appropriate therapy, while cases that evolve from a low-grade process may respond to therapy only to leave behind an incurable disease like fol- licular lymphoma. In addition, a common scenario in the stomach is the finding of DLBCL with an associated MALT FIGURE 8.49 This MALT lymphoma has an abundance lymphoma. Morphologically, MALT lymphomas may con- of somewhat larger cells and could be mistaken for a sist of cells that are large and have ample cytoplasm (Figure DLBCL at a cursory glance. Truly large, centroblast-like 8.49). Furthermore, scattered large cells are common in cells, however, are still individually scattered (arrow). 212 Neoplastic Gastrointestinal Pathology: An Illustrated Guide transferase (TdT). T cell lymphomas such as enteropathy- Carmack SW, Genta RM, Schuler CM, Saboorian MH. The current associated T cell lymphoma (see Chapter 9) and anaplastic spectrum of gastric polyps: a 1-year national study of over 120,000 patients. Am J Gastroenterol. 2009;104:1524–1532. large cell lymphoma may also resemble DLBCL, but their Carneiro F, David L, Seruca R, et al. Hyperplastic polyposis and lack of B cell marker expression by immunohistochemistry diffuse carcinoma of the stomach. A study of a family. Cancer. is helpful in most cases. 1993;72:323–329. The prognosis of DLBCL depends on the stage at pre- Gonzalez-Obeso E, Fujita H, Deshpande V, et al. Gastric hyperplastic polyps: a heterogeneous clinicopathologic group including a dis- sentation, including the involvement of regional lymph tinct subset best categorized as mucosal prolapse polyp. Am J Surg nodes. Like other aggressive lymphomas, it is potentially Pathol. 2011;35:670–677. curable, though an underlying low-grade component Hattori T. Morphological range of hyperplastic polyps and carcino- should be recognized and mentioned in the diagnosis since mas arising in hyperplastic polyps of the stomach. J Clin Pathol. 1985;38:622–630. it may persist even after the DLBCL is treated. Standard Niv Y, Delpre G, Sperber AD, et al. Hyperplastic gastric polyposis, therapy for DLBCL has long been the “CHOP” regimen, hypergastrinaemia, and colorectal neoplasia: a description of four consisting of cyclophosphamide, vincristine, doxorubicin, cases. Eur J Gastroenterol Hepatol. 2003;15:1361–1366. and dexamethasone. More recently, anti-CD20 immu- Ogata H, Oshio T, Ishibashi H, et al. Heterotopic pancreas in children: review of the literature and report of 12 cases. Pediatr Surg Int. notherapy with rituximab has been added successfully 2008;24:271–275. (R-CHOP) and is now the standard of care. Interestingly, Rich A, Toro TZ, Tanksley J, et al. Distinguishing Menetrier’s disease some targeted therapies aimed at BCL2 inhibitors (in the from its mimics. Gut. 2010;59:1617–1624. case of germinal center B cell phenotype) and the NF-κB Triffin A, Tarcoveanu E, Danciu M, et al. Gastric heterotopic pancreas: an unusual case and review of the literature. J Gastrointest Liver pathway (in the case of activated B cell phenotype) are Dis. 2012;21:209–212. under investigation. Additionally, there are some reports Wei R, Want QB, Chen QH, et al. Upper gastrointestinal tract hetero- of DLBCLs of limited stage in the stomach that are associ- topic pancreas: findings from CT and endoscopic imaging with his- ated with underlying H. pylori infection being amenable to topathologic correlation. Clin Imaging. 2011;35:353–359. Zea-Iriarte WL, Sekine I, Itsuno M, et al. Carcinoma in gastric hyper- very conservative therapy aimed at H. pylori eradication. plastic polyps. A phenotypic study. Dig Dis Sci. 1996;41:377–386. In aggressive cases with large ulcers or intractable bleed- ing, surgical therapy may be required, though surgery is not generally considered front-line therapy for lymphoma. Hamartomatous Polyps Amason T, Liang WY, Alfaro E, et al. Morphology and natural history of familial adenomatous polyposis-associated dysplastic fundic SELECTED REFERENCES gland polyps. Histopathology. 2014;65:353–362. Bettington M, Brown IS, Kumarasinghe MP, et al. The challenging diagnosis of Cronkhite-Canada syndrome in the upper gastroin- General testinal tract: a series of 7 cases with clinical follow-up. Am J Surg Carneiro F, Lauwers GY. Epithelial tumors of the stomach. In: Shepherd Pathol. 2014;38:215–233. NA, Warren BF, et al, eds. Morson and Dawson’s Gastrointestinal Bianchi LK, Burke CA, Bennett AE, et al. Fundic gland polyp dysplasia Pathology. 5th ed. West Sussex: Wiley-Blackwell; 2013. is common in familial adenomatous polyposis. Clin Gastroenterol Chun N, Ford JM. Genetic testing by cancer site: stomach. Cancer J. Hepatol. 2008;6:180–185. 2012;18:355–363. Burke AP, Sobin LH. The pathology of Cronkhite-Canada polyps. A com- Correa P. Human

gastric carcinogenesis: a multistep and multifactorial parison to juvenile polyposis. Am J Surg Pathol. 1989;13:940–946. process. Cancer Res. 1992;52:6735–6740. Burt RW. Gastric fundic gland polyps. Gastroenterology. Lauwers GY. Epithelial Neoplasms of the stomach. In: Odze RD, 2003;125:1462–1469. Goldblum JR, eds. Surgical Pathology of the GI Tract, Liver, Biliary Choudhry U, Boyce HW Jr., Coppola D. Proton pump inhibitor-asso- Tract, and Pancreas. 3rd ed. Philadelphia, PA: Elsevier; 2015. ciated gastric polyps: a retrospective analysis of their frequency, Naymagon S, Warner RR, Patel K, et al. Gastroduodenal ulceration and endoscopic, histologic, and ultrastructural characteristics. Am associated with radioembolization for the treatment of hepatic J Clin Pathol. 1998;110:615–621. tumors: an institutional experience and review of the literature. Chow E, Macrae F. A review of juvenile polyposis syndrome. J Dig Dis Sci. 2010;55:2450–2458. Gastroenterol Hepatol. 2005;20:1634–1640. Park DY, Lauwers GY. Gastric polyps: classification and management. Giardiello FM, Brensinger JD, Tersmette AC, et al. Very high risk of Arch Pathol Lab Med. 2008;132:633–640. cancer in familial Peutz-Jeghers syndrome. Gastroenterology. Syngal S, Brand RE, Church JM, et al. ACG clinical guideline: genetic 2000;119;1447–1453. testing and management of hereditary gastrointestinal cancer syn- Hizawa K, Iida M, Yao T, et al. Juvenile polyposis of the stomach: clini- dromes. Am J Gastroenterol. 2015;110;223–262. copathologic features and its malignant potential. J Clin Pathol. Turner JR, Odze RD. Polyps of the stomach. In: Odze RD, Goldblum 1997;50:771–774. JR, eds. Surgical Pathology of the GI Tract, Liver, Biliary Tract, Lam-Himlin D, Park JY, Cornish TC, et al. Morphologic char- and Pancreas. 3rd ed. Philadelphia, PA: Elsevier, 2015. acterization of syndromic gastric polyps. Am J Surg Pathol. 2010;34:1656–1662. Latchford AR, Neale K, Phillips RK, Clark SK. Juvenile polyposis syn- Infl ammatory Polyps, Hyperplastic drome: a study of genotype, phenotype, and long-term outcome. Polyps, and Non-neoplastic Lesions Dis Colon Rectum. 2012;55(10):1038–1043. Ma C, Giardiello FM, Montgomery EA. Upper tract juvenile polyps Abraham SC, Singh VK, Yardley JH, Wu TT. Hyperplastic polyps of in juvenile polyposis patients: dysplasia and malignancy are asso- the stomach: associations with histologic patterns of gastritis and ciated with foveolar, intestinal, and pyloric differentiation. Am J gastric atrophy. Am J Surg Pathol. 2001;25:500–507. Surg Pathol. 2014;38(12):1618–1626. 8 Neoplasms of the Stomach 213 McAllister AJ, Richards KF. Peutz-Jeghers syndrome: experience with You WC, Zhang L, Chang YS, et al. Gastric dysplasia and gastric can- twenty patients in five generations. Am J Surg. 1977;134:717–720. cer: Helicobacter pylori, serum vitamin C, and other risk factors. Odze RD, Marcial MA, Antonioli D. Gastric fundic gland polyps: J Natl Cancer Inst. 2000;92:1607–1612. a morphological study including mucin histochemistry, ste- reometry, and MIB-1 immunohistochemistry. Hum Pathol. 1996;27:896–903. Pyloric Gland Adenoma Pintiliciuc OG, Heresbach D, de-Lajarte-Thirouard AS, et al. Gastric Chen ZM, Scudiere JR, Abraham SC, Montgomery E. Pyloric gland ade- involvement in juvenile polyposis associated with germline noma. An entity distinct from gastric foveolar type adenoma. Am J SMAD4 mutations: an entity characterized by a mixed hypertro- Surg Pathol. 2009;33:186–193. phic and polypoid gastropathy. Gastroenterol Clin Biol. 2008;32 Kushima R, Ruthlein HJ, Stolte M, et al. Pyloric gland-type adenoma aris- (5 Pt 1): 445–450. ing in heterotopic gastric mucosa of the duodenum with dysplastic Swwetser S, Ahlquist DA, Osborn NK, et al. Clinicopathologic features progression of the gastric type. Virchows Arch. 1999;435:452–457. and treatment outcomes in Crokhite-Canada syndrome: support Vieth M, Kushima R, Borchard F, Stolte M. Pyloric gland adenoma: a clinico- for autoimmunity. Dig Dis Sci. 2012;57:496–502. pathological analysis of 90 cases. Virchows Arch. 2003;442:317–321. Torbenson M, Lee HH, Cruz-Correa M, et al. Sporadic fundic gland Vieth M, Montgomery EA. Some observations on pyloric gland ade- polyposis: a clinical, histological, and molecular analysis. Mod noma: an uncommon and long ignored entity! J Clin Pathol. Pathol. 2002;15:718–723. 2014;67:883–890. Van Lier MG, Wagner A, Mathus-Vliegen EM, et al. High cancer risk in Peutz-Jeghers syndrome: a systematic review and surveillance recommendations. Am J Gastroenterol. 2010;105:1258–1264. Oxyntic Gland Polyp/Adenoma Zacharin M, Bajpai A, Chow CW, et al. Gastrointestinal polyps in McCune Albright syndrome. J Med Genet. 2011;48:458–461. Singhi AD, Lazenby AJ, Montgomery EA. Gastric adenocarcinoma with Zelter A, Fernandez JL, Bilder C, et al. Fundic gland polyps and asso- chief cell differentiation: a proposal for reclassification as oxyntic ciation with proton pump inhibitor intake: a prospective study in gland polyp/adenoma. Am J Surg Pathol. 2012;36:1030–1035. 1780 endoscopies. Dig Dis Sci. 2011;56:1743–1748. Ueyama H, Yao T, Nakashima Y, et al. Gastric adenocarcinoma of fundic gland type (chief cell predominant type): proposal for a new entity of gastric adenocarcinoma. Am J Surg Pathol. Gastric Adenomas and Flat Dysplasia 2010;34:609–619. Abraham Sc, Montgomery EA, Singh VK, et al. Gastric adenomas: intestinal-type and gastric-type adenomas differ in the risk of ade- Adenocarcinoma nocarcinoma and presence of background mucosal pathology. Am J Surg Pathol. 2002;26:1276–1285. Blot WJ, Devesa SS, Kneller RW, et al. Rising incidence of adenocarcinoma Baek DH, Kim GH, park do Y, et al. Gastric epithelial dysplasia: charac- of the esophagus and gastric cardia. JAMA. 1991;265:1287–1289. teristics and long-term follow-up results after endoscopic resection Bunt AM, Hermans J, Smit VT, et al. Surgical /pathologic-stage migra- according to morphological categorization. BMC Gastroenterol. tion confounds comparisons of gastric cancer survival rates between 2015;15:249. Japan and Western countries. J Clin Oncol. 1995;13:19–25. Bearzi I, Brancorsini D, Santinelli A, et al. Gastric dysplasia: a ten-year Campoli PM, Ejima FH, Cardoso DM, et al. Metastatic cancer to the follow up study. Pathol Res Pract. 1994;190:61–68. stomach. Gastric Cancer. 2006;9:19–25. Everett SM, Axon AT. Early gastric cancer in Europe. Gut. Cunningham SC, Kamangar F, Kim MP, et al. Survival after gastric 1997;41:142–150. adenocarcinoma resection: eighteen year experience at a single Farinati F, Rugge M, Di Mario F, et al. Early and advanced gastric institution. J Gastrointest Surg. 2005;9:718–725. cancer in the follow up of moderate and severe gastric dysplasia Dhingra S, Wang H. Nonneoplastic signet-ring cell change in gastroin- patients. A prospective study. I.G.G.E.D. Interdisciplinary Group testinal and biliary tracts: a pitfall for overdiagnosis. Ann Diagn on Gastric Epithelial Dysplasia. Endoscopy. 1993;25:261–264. Pathol. 2011;15:490–496,. Jang JS, Choi SR, Qureshi W, et al. Long-term outcomes of endo- Eckardt VF, Giessler W, Kanzler G, et al. Clinical and morphologi- scopic submucosal dissection in gastric neoplastic lesions at cal characteristics of early gastric cancer: a case-control study. a single institution in South Korea. Scand J Gastroenterol. Gastroenterol. 1990;98:708–714. 2009;44:1315–1322. Esaki Y, Hirayama R, Hirokawa K. A comparison of patterns of Lauwers GY. Defining the pathology diagnosis of metaplasia, atro- metastasis in gastric cancer by histologic type and age. Cancer. phy, dysplasia, and gastric adenocarcinoma. J Clin Gastroenterol. 1990;65:2086–2090. 2003;36:S37–S43. Goseki N, Takizawa T, Koike M. Differences in the mode of the exten- Park DY, Srivastava A, Kim GH, et al. Adenomatous and foveolar sion of gastric cancer classified by histological type: new histologi- gastric dysplasia: distinct patterns of mucin expression and back- cal classification of gastric carcinoma. Gut. 1991;33:606–612. ground intestinal metaplasia. Am J Surg Pathol. 2008;32:524–533. Green LK. Hematogenous metastases to the stomach: a review of 67 Rugge M, Capelle LG, Cappellesso R, et al. Precancerous lesions in cases. Cancer. 1990;65:1596–1600. the stomach: from biology to clinical patient management. Best Huang KH, Want RF, Yang MH, et al. Advanced gastric cancer patients Practice Res Clin Gastroenterol. 2013;27:205–223. with lymphoid stroma have better survival than those without. Salirao U, Lauwers GY, Vieth M, et al. Gastric high grade dysplasia can J Surg Oncol. 2013;107:523–528. be associated with submucosal invasion: evaluation of its preva- Hughes C, Greywoode G, Chetty R. Gastric pseudo-signet ring cells: lence in a series of 121 endoscopically resected specimens. Am J a potential diagnostic pitfall. Virchows Arch. 2011;459:347–349. Surg Pathol. 2014;38:1545–1550. Hundahl SA, Phillips JL, Menck HR. The National Cancer Data Base Schlemper RJ, Hirata I, Dixon MF. The macroscopic classification of Report on poor survival of U.S. gastric carcinoma patients treated early neoplasia of the digestive tract. Endoscopy. 2002;34:163–168. with gastrectomy: Fifth Edition American Joint Committee on Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of Cancer staging, proximal disease, and the “different disease” gastrointestinal epithelial neoplasia. Gut. 2000;47:251–255. hypothesis. Cancer. 2000;88:921–932. Yamada H, Ikegami M, Shimoda T, et al. Long term follow-up study of Ishikura H, Kirimoto K, Shamoto M, et al.: Hepatoid adenocarcinomas of gastric adenoma/dysplasia. Endoscopy. 2004;36:390–396. the stomach: an analysis of seven cases. Cancer. 1986;58:119–126. 214 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Lauren P. The two histological main types of gastric carcinoma: diffuse Hebbard PC, Macmillan A, Huntsman D, et al. Prophylactic total gas- and so-called intestinal-type carcinoma-an attempt at a histo-clinical trectomy (PTG) for hereditary diffuse gastric cancer (HDGC): classification. Acta Pathol Microbiol Scand. 1965;64:31–49. the Newfoundland experience with 23 patients. Ann Surg Oncol. Lee SJ, Sohn TS, Lee J, et al. Adjuvant chemoradiation with 5-fluoro- 2009;16:1890–1895. uracil/leucovorin versus S-1 in gastric cancer patients following D2 Kaurah P, MacMillan A, Boyd N, et al. Founder and recurrent CDH1 lymph node dissection surgery: a feasibility study. Anticancer Res. mutations in families with hereditary diffuse gastric cancer. 2014;34:6585–6591. JAMA. 2007; 297:2360. Maehara Y, Orita H, Okuyama T, et al. Predictors of lymph node Oliveira C, Moreira H, Seruca R, et al. Role of pathology in the identi- metastasis in early gastric cancer. Br J Surg. 1992;79:245–247. fication of hereditary diffuse gastric cancer: report of a Portuguese Meng XM, Zhou Y, Dang T, et al. Magnifying chromoendoscopy com- family. Virchows Arch. 2005;446:181–184. bined with immunohistochemical staining for early diagnosis of Pharoah PD, Guilford P, Caldas C, International Gastric Cancer gastric cancer. World J Gastroenterol. 2013;19:404–410. Linkage Consortium. Incidence of gastric cancer and breast cancer Mezhir JJ, Gonen M, Ammori JB, et al. Treatment and outcome of in CDH1 (E-cadherin) mutation carriers from hereditary diffuse patients with gastric remnant cancer after resection for peptic ulcer gastric cancer families. Gastroenterology. 2001;121:1348–1353. disease. Ann Surg Oncol. 2011;18:670–676. Mori M, Iwashita A, Enjoji M. Adenosquamous carcinoma of the stomach: a clinicopathologic analysis of 28 cases. Cancer. 1986;57:333–339. Neuroendocrine Neoplasms Motoyama T, Aizawa K, Watanabe H, et al. α-Fetoprotein producing (See Also Chapter 3) gastric carcinomas: a comparative study of three different sub- types. Acta Pathol Jpn. 1993;43:654–661. Bordi C, Yu JY, Baggi MT, et al. Gastric carcinoids and their precursor Noda M, Kodama T, Atsumi M, et al. Possibilities and limitations lesions. A histologic and immunohistochemical study of 23 cases. of endoscopic resection for early gastric cancer. Endoscopy. Cancer. 1991;67(3):663–672. 1997;29:361–365. Bordi C. Gastric carcinoids: an immunohistochemical and clinicopath- O’Connell FP, Wang HH, Odze RD, et al. Utility of immunohistochem- ologic study of 104 patients. Cancer. 1995;75(1):129–130. istry in distinguishing primary adenocarcinomas from metastatic Bordi C. Neuroendocrine pathology of the stomach: the Parma contri- breast carcinomas in the gastrointestinal tract. Arch Pathol Lab Med. bution. Endocr Pathol. 2014;25:171–180. 2005;129:338–347. Ishida M, Sekine S, Fukagawa T, et al. Neuroendocrine carcinoma of Oda I, Kondo H, Yamao T, et al. Metastatic tumors to the stomach: the stomach: morphologic and immunohistochemical characteris- analysis of 54 patients diagnosed at endoscopy and 347 autopsy tics and prognosis. Am J Surg Pathol. 2013;37:949–959. cases. Endoscopy. 2001;33:507–510. La Rosa S, Inzani F, Vanoli A, et al. Histologic characterization and Okabayashi T, Gotoda T, Kondo H, et al. Early carcinoma of the gas- improved prognostic evaluation of 209 gastric neuroendocrine tric cardia in Japan: is it different from that in the West? Cancer. neoplasms. Hum Pathol. 2011;42(10):1373–1384. 2000;89:2555–2559. Rindi G, Bordi C, Rappel S, et al. Gastric carcinoids and neuroendo- Osada M, Aishima S, Hirahashi M, et al. Combination of hepatocellu- crine carcinomas: pathogenesis, pathology, and behavior. World J lar markers is useful for prognostication in gastric hepatoid adeno- Surg. 1996;20(2):168–172. carcinoma. Hum Pathol. 2014;45:1243–1250. Thomas RM, Baybick JH, Elsayed AM, Sobin LH. Gastric carcinoids. Saigo PE, Brigati DJ, Sternberg SS, et al. Primary gastric choriocarcinoma: An immunohistochemical and clinicopathologic study of 104 an immunohistological study. Am J Surg Pathol. 1981;5:333–342. patients. Cancer. 1994;73(8):2053–2058. Suan ZX, Ueyama T, Yao T, Tsuneyoshi M. Time trends of early gas- Yao JC, Hassan M, Phan A, et al. One hundred years after “carcinoid:” tric carcinoma. A clinicopathologic analysis of 2846 cases. Cancer. epidemiology of and prognostic factors for neuroendocrine tumors in 1993;72:2889–2894. 35,825 cases in the United States. J Clin Oncol. 2008;26:3063–3072. Tamura W, Fukami N. Early gastric cancer and dysplasia. Gastrointestinal Endoscopy Clin N Amer. 2013;23:77–94. Mesenchymal Neoplasms Wang HH, Wu MS, Shun Ct, et al. Lymphoepithelioma-like carcinoma of the stomach: a subset of gastric carcinoma with distinct clinico- (See Also Chapter 5) pathological features

and high prevalence of Epstein-Barr infec- Doyle LA, Nelson D, Heinrich MC, et al. Loss of succinate dehydrogenase tion. Hepatogastroenterology. 1999;46:1214–1219. subunit B (SDHB) expression is limited to a distinctive subset of gas- Wang K, Weinrach D, Lal A, et al. Signet-ring cell change versus signet- tric wild-type gastrointestinal stromal tumors: a comprehensive gen- ring cell carcinoma: a comparative analysis. Am J Surg Pathol. otype-phenotype correlation study. Histopathol. 2012;61:801–809. 2003;27:1429–1433. Kang G, Park HJ, Kim JY, et al. Glomus tumor of the stomach: a clini- Yakirevich E, Resnick MB. Pathology of gastric cancer and its precur- copathologic analysis of 10 cases and review of the literature. Gut sor lesions. Gastroenterol Clin North Am. 2013;42:261–284. Liver. 2012;6:52–57. Yasuda K, Adachi y, Shiraishi N, et al. Papillary adenocarcinoma of the Lasota J, Dansonka-Mieszkowska A, Sobin LH, Miettinen M. A great stomach. Gastric Cancer. 2000;3:33–38. majority of GISTs with PDGFRA mutations represent gastric tumors Yasuda K, Shiraishi N, Suematsu T, et al. Rate of detection of lymph node of low or no malignant potential. Lab Invest. 2004;84:874–883. metastasis is correlated with the depth of submucosal invasion in Liu TC, Lin MT, Montgomery EA, Singhi AD. Inflammatory fibroid polyps of early stage gastric carcinoma. Cancer. 1999;85;2119–2123. the gastrointestinal tract: spectrum of clinical, morphologic, and immu- Ye MF, Tao F, Liu F, Sun AJ. Hepatoid adenocarcinoma of the stomach: nohistochemistry features. Am J Surg Pathol. 2013;37(4):586–592. a report of three cases. World J Gastroenterol. 2013;19:4437–4492. Miettinen M, Makhlouf HR, Sobin LH, Lasota J. Plexiform fibromyx- oma: a distinctive benign gastric antral neoplasm not to be confused Hereditary Diffuse Gastric Cancer with a myxoid GIST. Am J Surg Pathol. 2009;33:1624–1632. Miettinen M, Sobin LH, Lasota J, et al. Gastrointestinal stromal Fitzgerald RC, Hardwick R, Huntsman D, et al. Hereditary diffuse tumors of the stomach. A clinicopathologic, immunohistochemi- gastric cancer: updated consensus guidelines for clinical man- cal, and molecular genetic study of 1765 cases with long term fol- agement and directions for future research. J Med Genet. 2010; low up. Am J Surg Pathol. 2005;29:52–68. 47:436–444. Miettinen M, Want ZF, Sarloma-Rikala M, et al. Succinate dehydroge- Gayther SA, Gorringe KL, Ramus SJ, et al. Identification of germ-line nase-deficient GISTs: a clinicopathologic, immunohistochemical, E-cadherin mutations in gastric cancer families of European ori- and molecular genetic study of 66 gastric GISTs with predilection gin. Cancer Res. 1998;58:4086–4089. to young age. Am J Surg Pathol. 2011;35:1712–1721. 8 Neoplasms of the Stomach 215 Ozolek JA, Sasatomi E, Swalsky PA, et al. Inflammatory fibroid pol- Fischbach W. Gastric MALT lymphoma – update on diagnosis and treat- yps of the gastrointestinal tract: clinical, pathologic, and molec- ment. Best Pract Res Clin Gastroenterol. 2014;28:1069–1077. ular characteristics. Appl Immunohistochem Mol Morphol. Kuo SH, Yeh KH, Wu MS, et al. Helicobacter pylori eradication therapy 2004;12:59–66. is effective in the treatment of early-stage H. pylori-positive gastric Takahashi Y, Shimizu S, Ishida T, et al. Plexiform angiomyx- diffuse large B-cell lymphomas. Blood. 2012;119:4838–4844. oid myofibroblastic tumor of the stomach. Am J Surg Pathol. O’Malley DP, Goldstein NS, Banks PM. The recognition and classi- 2007;31:724–728. fication of lymphoproliferative disorders of the gut. Hum Pathol. Voltaggio L, Murray R, Lasota J, Miettinen M. Gastric schwannoma: a 2014;45:899–916. clinicopathologic study of 51 cases and critical review of the litera- Owens SR, Smith LB. Molecular aspects of H. pylori-related ture. Hum Pathol. 2012;43:650–659. MALT lymphoma. Patholog Res Int. 2011;2011:193149. doi: 10.4061/2011/193149 Hematolymphoid Neoplasms Sehn LH, Gascoyne RD. Diffuse large B-cell lymphoma: optimiz- ing outcome in the context of clinical and biologic heterogeneity. Burke JS. Lymphoproliferative disorders of the gastrointestinal tract: a Blood. 2015;125:22–32. review and pragmatic guide to diagnosis. Arch Pathol Lab Med. Smith LB, Owens SR. Gastrointestinal lymphomas: entities and mimics. 2011;135:1283–1297. Arch Pathol Lab Med. 2012;136:865–870. 9 Neoplasms of the Small Intestine W EI CHEN, SCOT T R . OW ENS, AND W ENDY L . FR ANKEL INTRODUCTION Peptic duodenitis often appears nodular or erythematous at endoscopy; common histologic findings include reac- The small intestine represents 75% of the length and tive epithelial changes, gastric surface cell metaplasia, and greater than 90% of the surface area of the gastrointes- increased chronic (and sometimes acute) inflammation in tinal (GI) tract. Despite its length, large surface area, and the lamina propria (Figure 9.1A–C). Reactive epithelium higher rate of cellular turnover, primary small intestinal with foveolar metaplasia in nodular peptic duodenitis tumors are rare, estimated at 40 to 60 times less common can at times mimic dysplasia. Although many duodenal than colonic neoplasms, and account for only 3% of GI adenomas do have gastric surface cell metaplasia, adeno- neoplasms overall. Rapid transit time of the luminal liquid mas are more likely to be sharply delineated from adja- content, lower bacterial load, and detoxification effects of cent normal mucosa than reactive lesions (Figure 9.2A–B). mucosal enzymes are all believed to contribute to decreased Brunner gland hyperplasia is another common lesion that production of and/or exposure to carcinogens. The abun- often presents as a polypoid mass (Figure 9.3A–B). dant immunoglobulin A (IgA) and lymphoid tissue in the In the small bowel, gastric heterotopias most com- small bowel also enhances immunosurveillance for tumor. monly occur in the duodenal bulb and in Meckel’s diver- There are four major histologic categories of primary ticulum. These congenital rests are benign, non-neoplastic small intestinal malignancies: adenocarcinomas, neuroen- lesions composed of ectopic gastric mucosa containing both docrine tumors (NETs), mesenchymal tumors, and lym- foveolar epithelium and oxyntic glands (Figure 9.4A–B). phomas (Table 9.1). Epidemiologic studies have shown an Heterotopic gastric tissue should be distinguished from increased incidence of all types between 1985 and 2005, foveolar cell metaplasia of the duodenal mucosa, which with a more than four-fold increase in the incidence of is typically secondary to peptic injury, Crohn disease, or NETs. In fact, in the year 2000, NETs surpassed adeno- Helicobacter pylori infection. carcinoma as the most common type of primary small Pancreatic heterotopia (Figure 9.5A–B) occurs most intestinal tumor reported to the National Cancer Data often in the distal stomach and proximal small bowel, and Base. The World Health Organization (WHO) 2010 clas- is also common in Meckel’s diverticulum. Heterotopic pan- sification of small intestinal tumors is shown in Table 9.2. creas can be either mucosal or mural, with the latter often causing more significant clinical symptoms such as abdomi- nal pain, stricture, intussusception, or bleeding. One-third INFLAMMATORY AND NON-NEOPLASTIC of pancreatic heterotopia cases contain pancreatic islets, in LESIONS addition to lobules of pancreatic acini and ductules. Inflammatory pseudopolyps can be sporadic, or asso- Inflammatory lesions can present as polypoid masses, and ciated with inflammatory bowel disease, and may mimic thus lead to endoscopic biopsies or polypectomies. Several a neoplastic process endoscopically. Histologic examina- inflammatory and non-neoplastic entities enter into the tion reveals polypoid granulation tissue with inflamma- differential diagnosis of dysplasia and malignancy in tion, edema, and fibrosis, and there may be florid reactive the small bowel, and thus will be briefly discussed here. epithelial changes (Figure 9.6). 217 218 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 9.1 Clinicopathologic Characteristics of Primary Small Intestinal Malignancies Neuroendocrine Tumor Adenocarcinoma Lymphoma Mesenchymal Tumor Relative Incidence (%) 37.3 36.9 17.3 8.4 Age (median, years) 66 67 66 64 Gender (male: female) 1.1:1 1.1:1 1.4:1 1.1:1 Size (median, cm) 1.6 4.0 NA 7.5 Most common site Jejunum Duodenum Any site Any site Source: Data adapted from Bilimoria et al. Small bowel cancer in the United States: changes in epidemiology, treatment, and survival over the last 20 years. Ann Surg. 2009;249:63–71. TABLE 9.2 WHO 2010 Classification of Tumors of the HAMARTOMATOUS POLYPS Small Intestine Epithelial Tumors Several types of hamartomatous polyps occur in the small Premalignant Lesions bowel; these are also discussed in detail in Chapter 6. Adenoma Peutz–Jeghers polyps can occur sporadically or as part of Tubular Peutz–Jeghers syndrome (PJS). PJS is an inherited syndrome Villous Tubulovillous characterized by a germline mutation of the tumor suppres- Dysplasia (intraepithelial neoplasia) sor gene STK11/LKB1. The polyps preferentially affect the Low grade small intestine (most frequently the jejunum, followed by the High grade ileum and duodenum), and patients typically have muco- Hamartomas Juvenile polyp cutaneous melanocytic pigmentation. When compared to Peutz–Jeghers polyp the general population, PJS patients have a very high rela- Carcinoma tive risk (RR = 520) for the development of small intestinal Adenocarcinoma adenocarcinoma. Histologically, Peutz–Jeghers polyps show Mucinous adenocarcinoma (>50% mucinous) Signet ring cell carcinoma (>50% signet ring cells) papillary architecture with arborizing bundles of smooth Adenosquamous carcinoma muscle that separate the epithelial components into lobules Medullary carcinoma (Figure 9.7A–B). Dysplasia may occur within these polyps Squamous cell carcinoma (Figure 9.7C–D), and misplacement of both non-neoplastic Undifferentiated carcinoma Neuroendocrine Neoplasms and dysplastic epithelium is common in pedunculated pol- Neuroendocrine tumor (NET) yps. Displacement of dysplastic epithelium may mimic inva- NET G1 (carcinoid) sive cancer, but the identification of smooth muscle bundles NET G2 around the displaced epithelium, as opposed to a stromal Neuroendocrine carcinoma (NEC) Large cell NEC desmoplastic reaction, can help distinguish misplaced glands Small cell NEC from invasive adenocarcinoma (Figure 9.7E). Mixed adenoneuroendocrine carcinoma (MANEC) Juvenile polyps may also be either sporadic or syn- Enterochromaffin (EC) cell, serotonin-producing NET dromic. Sporadic juvenile polyps are restricted to the colorec- Gangliocytic paraganglioma Gastrinoma tum, and have no associated cancer risk. Juvenile polyposis L cell, glucagon-like peptide-producing and PP/PYY-producing syndrome (JPS) is characterized by a germline mutation in NETs the SMAD4 or BMPR1A gene, and multiple juvenile pol- Somatostatin-producing NET yps in the GI tract. These polyps are most commonly found Mesenchymal Tumors Leiomyoma in the colorectum, but can be found throughout the upper Lipoma and lower GI tract as well. JPS patients have a 68% risk Angiosarcoma of developing colorectal cancer by 60 years of age, with a Gastrointestinal stromal tumor mean age at diagnosis of 35 years. There is also increased Kaposi sarcoma Leiomyosarcoma risk for small intestinal, gastric, and possibly pancreatic Lymphomas cancers. Histologically, juvenile polyps show cystically Secondary Tumors dilated glands embedded in abundant loose, edematous and inflamed stroma (Figure 9.8A–B). Surface ulceration and Source: Data adapted from Bosman F, Carneiro F, Hruban R, et al (Eds.) Chapter 6, Tumours of the small intestine. In mucin extravasation from ruptured cysts may be present as World Health Organization clas- sification of tumours of the digestive system. 4th ed. Lyon, France:IARC Press; well. Syndromic juvenile polyps may give rise to foci of dys- 2010. plasia (Figure 9.8C–D) and carcinoma, but these findings are very uncommon in sporadic juvenile polyps. 9 Neoplasms of the Small Intestine 219 (A) (B) (C) FIGURE 9.1 Nodular peptic duodenitis features Brunner gland hyperplasia, surface gastric metaplasia, acute and chronic inflammation, and reactive epithelial changes (A–B). Reactive surface gastric cell metaplasia may occasionally mimic dysplasia, but the well-developed apical mucin is a clue that this is a reactive, metaplastic change rather than dysplasia (C). (A) (B) FIGURE 9.2 Small bowel adenomas are typically sharply demarcated from the surrounding mucosa, unlike reactive epithelial changes. The adenomatous epithelium on the left abruptly transitions to normal mucosa on the right (A–B). 2 2 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 9.3 Brunner gland hyperplasia can form a polypoid mass in the duodenum (A). Alcian blue/periodic acid–Schiff stain (B) highlights the goblet cells in the epithelium (blue) and Brunner glands in the submucosa and deep mucosa (magenta). (A) (B) FIGURE 9.4 Heterotopic gastric tissue in the small bowel occasionally forms polypoid masses (A). At higher power, the heterotopic tissue is composed of parietal and chief cells (B) admixed with the small bowel mucosa. (A) (B) FIGURE 9.5 Heterotopic pancreas in the submucosa of the small intestine is composed of a variable mixture of benign pancreatic acini, islets, and ductules (A–B, arrows denoting small islets). 9 Neoplasms of the Small Intestine 2 21 ADENOMATOUS POLYPS Small intestinal adenomas are polypoid growths of dys- plastic epithelium that are most often (80%) found near the ampulla/papilla of Vater or in the second portion of the duodenum. They may be incidental findings, or they can cause clinical signs and symptoms including GI bleed- ing, abdominal pain, intussusception, or jaundice, par- ticularly if located at the ampulla. Treatment options for small bowel adenomas include endoscopic polypectomy for pedunculated polyps, endoscopic mucosal or surgical resection for large sessile lesions, and Whipple surgery for large periampullary lesions. Most small intestinal adenomas resemble colonic adenomas both macroscopically and microscopically. One unique feature of

small intestinal adenomas is that many of them show gastric surface cell metaplasia, creating the illusion of surface maturation and thus mimicking a reac- tive process (Figure 9.9A). Small bowel adenomas tend to have a more villous or tubulovillous pattern (Figure 9.9B–C), as compared to the predominantly tubular pat- FIGURE 9.6 An ileal inflammatory pseudopolyp from a tern seen in colonic adenomas. Goblet cells are prevalent patient with Crohn disease, showing a polypoid projection in small bowel adenomas as well, and occasional Paneth of disordered mucosa protruding above the surrounding cells and endocrine cells may also be seen. As with colonic ulcerated mucosa. adenomas, dysplasia in small bowel adenomas is classified (A) (B) (C) (D) (E) FIGURE 9.7 A Peutz–Jeghers polyp of the duodenum features lobules of glandular epithelium separated by complex, arborizing bundles of smooth muscle (A–B). Peutz–Jeghers polyps may contain foci of dysplasia, as in this example with low-grade dysplasia at the surface (C). Note the arborizing muscle fibers that surround the dysplastic glands (D). Epithelial misplacement may pose a diagnostic problem in Peutz–Jeghers polyps, as noted here where dysplastic glands are misplaced within the bundles of smooth muscle in the stalk, mimicking invasive cancer (E). However, muscle bundles surround both the dysplastic glands and the nondysplastic glands, and there is no desmoplasia (B–E, courtesy of Dr. Samir Kahwash). 2 2 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 9.8 This small bowel polyp from a patient with juvenile polyposis syndrome shows cystically dilated glands embedded in abundant loose, edematous, and inflamed stroma (A–B). Some glands are filled with mucin and inflammatory debris. Dysplasia can be seen in syndromic juvenile polyps (C–D). In (C), notice the demarcation between the non- dysplastic (left) and dysplastic (right) areas. as either low or high grade. The risk of progression to Patients with Lynch syndrome have a 4% lifetime risk of adenocarcinomas in adenomas (Figure 9.9D–F) increases developing small bowel adenocarcinoma, which is 100 with increasing polyp size, with the presence of high- times greater than the risk in the general population. grade dysplasia, and with increasing percentage of villous MUTYH-associated polyposis is associated with an architecture in the polyp. increased risk for the development of duodenal adeno- Several hereditary cancer syndromes are associ- mas and adenocarcinomas as well. ated with small bowel adenomas, most notably familial adenomatous polyposis (FAP) and Lynch syndrome (see also Chapter 6). FAP is an autosomal dominant disorder ADENOCARCINOMA caused by a germline mutation of the adenomatous pol- yposis coli (APC) gene on chromosome 5q21. The life- Epidemiology time risk for FAP patients to develop duodenal adenomas and small bowel adenocarcinoma is estimated at nearly Primary small bowel adenocarcinoma is rare, with only 100% and 5%, respectively. Periampullary adenomas approximately 3,000 cases diagnosed per year. This is a appear to have a high risk of malignant transformation. 50-fold lower incidence than colorectal adenocarcinoma, 9 Neoplasms of the Small Intestine 2 2 3 (A) (B) (C) (D) (E) (F) FIGURE 9.9 Small bowel adenomas are in some cases similar to their colonic counterparts (A), but often have a more prominent villous component (B–C). Note the gastric surface cell metaplasia in this duodenal adenoma as well (A, arrow). This jejunal adenoma has a focus of invasive adenocarcinoma in the stalk (D–E). Higher power shows infiltrating irregular glands with associated desmoplasia (F). 2 2 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide despite the fact that the small intestine represents 75% of the Additionally, adenocarcinomas rarely arise in heterotopic length and 90% of the surface area of the alimentary tract. tissues in the small bowel, such as heterotopic pancreas The mechanisms of carcinogenesis are less well- and Meckel’s diverticulum. defined in the small intestine than in the large intestine. The adenoma–adenocarcinoma sequence is assumed to be Clinical and Prognostic Features similar, however, and 80% of duodenal adenocarcinomas The most common location for sporadic primary small have an associated adenomatous component. Patients with bowel adenocarcinomas is in the duodenum (55%, espe- polyposis syndromes, such as FAP, Lynch syndrome, PJS, cially at the ampulla of Vater), followed by jejunum (30%) JPS, and PTEN hamartoma tumor syndrome (PHTS), and ileum (15%). In contrast, adenocarcinomas arising in have increased risk of small bowel malignancies. Chronic the setting of celiac disease and Crohn disease preferen- inflammation is another major risk factor associated with tially occur in the jejunum and distal ileum, respectively, the development of small bowel adenocarcinoma, as dem- likely corresponding to the site of the most significant onstrated by the increased incidence of small intestinal chronic mucosal injury. adenocarcinoma in patients with Crohn disease, celiac The diagnosis of small bowel adenocarcinoma is often disease, and ileostomies. Other risk factors for small made at a more advanced stage (28% and 32% at stages bowel adenocarcinoma include cigarette smoking, alcohol III and IV, respectively) than colorectal cancer (27% and consumption, and toxin exposure to 7,12-dimethylbenz- 20% at stages III and IV, respectively), most likely due anthracene (DMBA) and benzopyrene. A summary of to the relative inaccessibility of some parts of the small diseases associated with the development of small bowel bowel, nonspecific presenting symptoms, and low index adenocarcinoma is given in Table 9.3. of clinical suspicion. In a large population-based compari- Small bowel adenocarcinoma occasionally arises in son of adenocarcinomas of small and large intestine, small the background of restorative proctocolectomy with ileal bowel adenocarcinoma patients were younger, presented pouch. When the small bowel mucosa in ileal pouches at a higher stage and with a higher histological tumor adapts to its role as a reservoir, the mucosa often under- grade, and had a worse outcome than those with colorec- goes villous atrophy, and pouchitis may develop. Dysplasia tal adenocarcinoma (Table 9.4). and adenocarcinoma can occur in this background of Overall, the prognosis for small bowel adenocarcino- chronic inflammation, but this is a very rare occurrence. mas is poor, with a 5-year relative survival of less than TABLE 9.3 Diseases Associated With Small Intestinal Adenocarcinoma FAP Lynch Syndrome Peutz–Jeghers Syndrome Crohn Disease Celiac Disease Molecular alterations APC (5q21) Mismatch repair STK11/LKB1 NA NA genes (19p13.3) Premalignant lesions Adenomas Adenomas Hamartomas with dysplasia Epithelial Epithelial and adenomas dysplasia dysplasia associated associated with chronic with chronic inflammation inflammation Location of polyps Colon, duodenum/ Colon, duodenum, Small intestine, stomach, Ileum Jejunum periampulla, jejunum, ileum colon jejunum, and ileum Risk for develop- Lifetime risk Relative risk 13% by age 65 years Relative risk Relative risk ing small intestinal 3%–5% >100 33.2 60–80 adenocarcinoma Lifetime risk 2%–8% Recommendations Gastro-duodenal No established Upper endoscopy or video No established No established for small bowel endoscopy guidelines capsule endoscopy every guidelines guidelines surveillance every 1–5 years 2–3 years from 18 years depending on of age polyp location and burden Abbreviation: FAP, familial adenomatous polyposis. Source: Data from Arber and Moshkowitz. Small bowel polyposis syndromes. Curr Gastroenterol Rep. 2011;13:435–441; Canavan et al. Meta-analysis: colorec- tal and small bowel cancer risk in patients with Crohn’s disease. Aliment Pharmacol Ther. 2006;23:1097–1104; Jass. Colorectal polyposes: from phenotype to diagnosis. Pathol Res Pract. 2008;204:431–447; Koornstra. Small bowel endoscopy in familial adenomatous polyposis and Lynch syndrome. Best Pract Res Clin Gastroenterol. 2012;26:359–368; Pan. Epidemiology of cancer of the small intestine. World J Gastrointest Oncol. 2011;3:33–42; Rampertab et al. Small bowel neoplasia in coeliac disease. Gut. 2003;52:1211–1214. 9 Neoplasms of the Small Intestine 2 2 5 TABLE 9.4 Comparison of Small and Large Bowel Adenocarcinomas Small Bowel Adenocarcinoma Large Bowel Adenocarcinoma Age Younger Older Gender 54% Male 49% Male Race 16% Black 10% Black Age-standardized incidence rate Decreased (−1.24%/year) Increased (+1.47%/year) High-grade tumor 33% 21% Stage IV at presentation 32% 20% Five-year cancer-specific survival Stage I: 13% worse Better (among patients with ≥ 8 lymph nodes Stage II: 15.9% worse Better assessed) Stage III: 18.5% worse Better Stage IV: No difference No difference Molecular alterations 7%–13% 60%–68% APC mutation 47% 73% 18q- 40%–60% 40%–60% KRAS mutation 18%–35% (67%–73% in Celiac-related 15% Microsatellite small bowel adenocarcinoma) Instability Source: Data adapted from Overman et al. A population-based comparison of adenocarcinoma of the large and small intestine: insights into a rare disease. Ann Surg Oncol. 2012;19:1439–1445; Raghav and Overman 2013. Small bowel adenocarcinomas—existing evidence and evolving paradigms. Nat Rev Clin Oncol. 2013;10:534–544. 30%. Other small bowel neoplasms, such as NETs and (15%, vs. colon 8%), and CK7+/CK20- (13%, vs. colon lymphomas, generally have a better prognosis than adeno- <1%). CDX2 may be expressed, but less frequently than carcinoma. Surgical resection is the treatment of choice in the colon. Tumors arising at the ampulla of Vater may for small bowel adenocarcinoma. The role of adjuvant show either intestinal or pancreaticobiliary-type morphol- therapy remains unclear, and mucin-producing adenocar- ogy as well as immunophenotype; the intestinal type is cinomas are typically resistant to radiation therapy. associated with a better prognosis. Not surprisingly, small and large bowel adeno- Macroscopic and Microscopic Features carcinomas share some similar molecular alterations, such as 18q loss (less common in the small bowel than Macroscopically, most small bowel adenocarcinomas are colon), TP53 overexpression, and activating mutations annular constricting tumors, but they may also present in KRAS. Small bowel adenocarcinomas demonstrate as flat, polypoid, or ulcerative lesions. Duodenal carcino- similar rates of microsatellite instability (15 to 20%) and mas are usually more circumscribed and polypoid, due to CpG island methylator phenotype (27%) to those seen frequent association with an adenomatous component. in colorectal cancer. However, celiac-disease-related Microscopically, small bowel adenocarcinomas resemble small bowel adenocarcinomas show higher rates of MSI their counterparts in the colorectum (Figure 9.10A–D). (67%–73%) due to promoter methylation of MLH1. Small bowel adenocarcinomas associated with Crohn dis- Given the comparable rates of MSI in small and large ease resemble sporadic adenocarcinomas as well, but are bowel adenocarcinomas, we suggest incorporating the more likely to have poorly differentiated or mucinous results of mismatch repair protein immunostains or MSI features. testing on primary small bowel adenocarcinomas into Rare subtypes of small bowel carcinoma include ade- surgical pathology reports. nosquamous carcinoma (Figure 9.10E–F), medullary carci- Interestingly, APC mutation occurs at a much lower noma, mixed adenocarcinoma/neuroendocrine carcinoma rate in small bowel adenocarcinoma (7%–13%) than in (NEC), tumors with tripartite differentiation (glandular, tumors of the large bowel (60%–68%), which may par- squamous, and neuroendocrine components), and small tially explain the lower number of adenomas and con- cell NECs. sequently fewer adenocarcinomas observed in the small bowel. Ancillary Studies Forty-three percent of small bowel adenocarcinomas are CK7-/CK20+, as opposed to 82% of colorectal adenocar- Diagnostic Challenges cinomas. Twenty-eight percent are CK7-/CK20- (as com- Adenomas with high-grade dysplasia can be difficult to pared to 10% in the colon), followed by CK7+/CK20+ differentiate from invasive adenocarcinoma, particularly 2 2 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 9.10 This case of primary duodenal signet ring cell adenocarcinoma is composed of infiltrating dyscohesive cells with eccentric, crescent-shaped, hyperchromatic nuclei displaced by cytoplasmic mucin (A–B). This ileal adenocarcinoma arising in the context of Crohn disease shows infiltrating small, irregular malignant glands with overlying ulceration (C–D). An ileal adenosquamous carcinoma shows neoplastic glands admixed with malignant squamoid cells with keratin formation (E–F). 9 Neoplasms of the Small Intestine 2 27 in small biopsies, in specimens obtained from around the in the ileum) and as previously discussed in the section on ampulla due to the complex architecture in this location, non-neoplastic lesions, ectopic pancreas (more common or in adenomas with prolapse change/misplaced epithe- in proximal than distal small intestine). The presence lium. Stromal desmoplasia and the presence of more severe of endometrial glands (especially the ciliated epithelium atypia in the putative invasive component than in the sur- associated with tubal metaplasia), hypercellular endo- face adenomatous epithelium can be helpful in making the metrial stroma, and hemosiderin supports the diagnosis diagnosis of invasion (Figure 9.11A–D). Pancreatic adeno- of endometriosis. Positive staining with estrogen recep- carcinomas in the ampullary/periampullary area can also tor (ER), PAX8, and CD10 and negative CDX2 are help- mimic small bowel adenocarcinoma, as these tumors may ful in confirming the diagnosis of endometriosis as well. extend outwards from the ampulla and grow along the Ectopic pancreas may also be misinterpreted as adenocar- basement membrane of the small bowel epithelium, thus cinoma, particularly on frozen section evaluation when simulating a small bowel adenocarcinoma arising from an only pancreatic ducts are present (Figure 9.12A–B). The adenoma. lobulated architecture and lack of

cytologic atypia help Several benign conditions can mimic small bowel ade- to support a diagnosis of ectopic pancreas rather than nocarcinoma, including endometriosis (most commonly adenocarcinoma. (A) (B) (C) (D) FIGURE 9.11 This low-power photomicrograph shows invasive adenocarcinoma arising from the base of a jejunal tubulovillous adenoma with high-grade dysplasia (A). When compared to the adenomatous epithelium at the surface (B), the invasive glands demonstrate more severe atypia with vesicular nuclei, open chromatin, prominent nucleoli, and single cell necrosis (C). There is also prominent desmoplasia around the invasive component (D). 2 2 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 9.12 Ectopic pancreas may contain only ducts, without accompanying acini and islets, mimicking invasive adenocarcinoma in the duodenal submucosa (A–B). However, there is retained lobular architecture surrounding smooth muscle, and a lack of cytological atypia. Grading and Staging Metastases in one to three regional nodes are clas- sified as pN1, and metastases to four or more nodes The current grading and staging criteria for small bowel are classified as pN2. pM1 indicates distant metastasis. adenocarcinomas are based on AJCC/UICC TNM, sev- Notably, due to the rich network of lymphatic vessels in enth edition. Grading of small bowel adenocarcinoma is the small bowel lamina propria, carcinomas invading similar to that of colorectal cancer, and is summarized in the lamina propria of small bowel are staged as pT1a, brief as follows: whereas similar lesions are staged as in situ disease (pTis) • Gx: grade cannot be assessed in the colon. • G1: well-differentiated • G2: moderately differentiated • G3: poorly differentiated NEUROENDOCRINE NEOPLASMS • G4: small cell carcinoma and undifferentiated carcinoma Epidemiology and Clinicopathologic Features By convention, signet ring cell carcinoma is grade 3. Most The majority of GI well-differentiated NETs arise in the small bowel adenocarcinomas are moderately (53%) or small intestine (45%), followed by rectum (20%), appen- well differentiated (23%). Overall, 21% are classified as dix (16%), colon (11%), and stomach (7%). Small bowel poorly differentiated, and 2% as undifferentiated carci- NETs occur most frequently in the ileum (specifically nomas. Grade does not appear to be a strong predictor of within 60 cm of the ileocecal valve), followed by duode- outcome. num (especially in the first and the second parts), and then Staging criteria for small intestinal adenocarcinoma jejunum. are listed as follows: The types of small bowel NET parallel the progenitor • pT1: tumor invades lamina propria or submucosa cell types in the foregut and midgut, and different NETs • pT2: tumor invades muscularis propria characteristically present with distinct clinical symptoms. • pT3: tumor invades through the muscularis propria Midgut NETs, including jejunal and ileal tumors, are into the subserosa or into the nonperitonealized enterochromaffin (EC) cell tumors, which produce sero- perimuscular tissue (mesentery or retroperitoneum) tonin and other vasoactive substances. When liver metas- with extension 2 cm or less tases are present, they cause classic carcinoid syndrome • pT4: tumor penetrates visceral peritoneum (serosa) (flushing and diarrhea) and frequent fibrous endocardial or invades other organs or structures (including thickening on the right side of the heart. Twenty percent other loops of small intestine, mesentery, or retro- of these tumors are multifocal, and they are not associated peritoneum greater than 2 cm, and abdominal wall with hereditary tumor syndromes. Foregut NETs, includ- by way of serosa; for duodenum only, invasion of ing duodenum and proximal jejunum, comprise gastrin- pancreas or bile duct) secreting G-cell tumors, somatostatin-producing D-cell 9 Neoplasms of the Small Intestine 2 2 9 tumors, and rarely EC-cell tumors. One third of gastri- gray in the fresh state, and turn yellow following forma- nomas are associated with Zollinger–Ellison syndrome lin fixation (Figure 9.13A–B). The overlying mucosa may (hypergastrinemia, gastric hypersecretion, and refractory be intact or ulcerated. In general, the tumor size is small peptic-ulcer disease). Gastrinomas are also associated (less than 2 cm), and even smaller in functioning/hormone with multiple endocrine neoplasia type 1 (MEN1) syn- secreting NETs (eg, gastrinomas are typically less than 0.8 drome. Small (less than 1.0 cm) nonfunctional gastrino- cm, and somatostatinomas average 1.8 cm). In some cases, mas are often associated with chronic H. pylori gastritis deep infiltration into the muscular wall, peritoneum, or and long-term proton pump inhibitor use. In contrast mesentery is present, which can lead to obstruction, adhe- to pancreatic somatostatin-producing NETs, duodenal sion, and volvulus. somatostatinomas are not associated with “somatostati- A detailed description of the histologic features of noma syndrome” (diabetes mellitus, cholelithiasis, and NETs is given in Chapter 3. Briefly, low-grade NET con- steatorrhea). However, duodenal somatostatinomas are tains uniform round smooth nuclei with stippled chroma- associated with MEN1, neurofibromatosis type 1 (NF1), tin and inconspicuous nucleoli (Figure 9.13C–E). Tumor and von Hippel–Lindau syndrome (VHL). Notably, L-cell, cells may be arranged in various patterns, including nested/ glucagon-like peptide-producing, and pancreatic peptide/ insular, trabecular, glandular/pseudoglandular, or solid. peptide YY-producing NETs are much less common in the NETs are typically very vascular, with frequent perineural small bowel (foregut and midgut) than the hindgut (left and vascular invasion even in well-differentiated tumors. colon and rectum). NETs are discussed in greater detail Well-differentiated small bowel NETs are positive for in Chapter 3. neuroendocrine markers (synaptophysin, chromogranin, Many small bowel NETs are found incidentally, and and CD56) and cytokeratin (CK) AE1/3, and negative are asymptomatic at presentation due to hepatic metab- for mucin, TTF1, and S100. Poorly differentiated small olism of vasoactive amines, absence of hormone secre- bowel NECs are uncommon, but similar to other poorly tion altogether, and/or small size. Among symptomatic differentiated NETs in that they can stain for TTF1 and patients, 40% present with vague and nonspecific abdom- show weak to absent expression of chromogranin and CK inal pain, secondary to intussusception, mass effect of the AE1/3. tumor, or mesenteric ischemia due to tumor-associated Gangliocytic paraganglioma is a rare nonfunctional, fibrosis. Intermittent bowel obstruction occurs in 25% of triphasic tumor that primarily occurs in the second por- small intestinal NET, and duodenal/periampullary NET tion of duodenum, and occasionally in the pylorus and may also produce biliary obstruction. jejunum. This tumor is generally regarded as benign, Small bowel NETs have a propensity to metastasize to with rare reports of regional lymph node metastases. the liver, mesentery, and peritoneum. The metastatic risk Macroscopically, gangliocytic paraganglioma is a small of small bowel NET increases when tumors are greater submucosal mass that frequently has an ulcerated muco- than 2.0 cm, when the muscularis propria is involved, and sal surface. It is composed of variable proportions of with increased mitotic activity. The prognosis for small spindle cells, epithelioid cells, and ganglion-like cells bowel well-differentiated NET is worse than for those (Figure 9.14A–D). The spindle cells resemble prolifer- arising in the stomach or rectum. Treatment modalities ating neural processes and Schwann cells; the epithe- include surgery, somatostatin analogues (if the tumor lioid cells are arranged in an “endocrine” pattern that expresses somatostatin receptors), and chemoradiation. forms nests or trabeculae; and the ganglion-like cells The latter has limited utility in treating well-differentiated are large cells with round nuclei and distinct nucleoli. NET. Immunohistochemically, the spindle cell component is positive for S100; the epithelioid component stains for Molecular Features synaptophysin, chromogranin, and occasionally CK AE1/3 (50%); and the ganglion cells express synaptophy- Molecular features of foregut NETs include frequent sin. The major differential diagnosis on hematoxylin and abnormalities of the MEN1 gene, which is absent in mid- eosin (H&E) evaluation includes GISTs (which are posi- gut (ileal) NETs. Ileal NETs often show mutations in the tive for KIT), well- differentiated NETs, and ganglioneu- APC gene and upregulation of the HOXC6 gene. In addi- romas. Identification of all three cell components helps tion, several growth factors, such as transforming growth to distinguish gangliocytic paraganglioma from the other factor (TGF), insulin-like growth factor (IGF), and fibro- entities in the differential diagnosis. blast growth factor (FGF), play important roles in the stromal fibrosis/desmoplasia seen in EC-cell NETs. Diagnostic Challenges Macroscopic and Microscopic Features Somatostatinomas may show a purely glandular pattern that can be confused with conventional adenocarcino- Macroscopically, small bowel NETs can be solitary or mas. Immunohistochemical stains for neuroendocrine multiple (25%–40% cases). They are characteristically markers are useful in such cases. It is also important to 2 3 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 9.13 This well-circumscribed, round, pink, well-differentiated neuroendocrine tumor bulges into the lumen of the duodenum (A, courtesy Dr. Whit Knapple). Neuroendocrine tumors characteristically turn yellow after formalin fixation (B). An example of the typical nested or organoid growth pattern of a well-differentiated NET (C) composed of uniform round nuclei with stippled chromatin and inconspicuous nucleoli (D). Diffuse strong synaptophysin staining is typical (E). recognize that some NETs produce mucin or have spindle Grading and Staging cell components, and the majority of NETs will show Grading of small bowel NET is performed similarly to CK expression. Some well-differentiated NETs that are other NETs, which is based on mitotic rate and Ki-67 pro- arranged in sheets may mimic lymphoma, but lymphomas liferative index (WHO 2010 criteria). This is discussed in express CD45, rather than epithelial and neuroendocrine detail in Chapter 3. Accurate grading is difficult on small markers. biopsies, given the limited sample, and should be performed 9 Neoplasms of the Small Intestine 2 31 (A) (B) (C) (D) FIGURE 9.14 Duodenal gangliocytic paragangliomas are typically well-circumscribed tumors located in the submucosa (A). They are composed of a mixture of epithelioid cells, spindle cells, and ganglion cells (B). These lesions strongly and diffusely express neuroendocrine markers such as chromogranin and synaptophysin (C), and S100 highlights the sustentacular network (D). on resection specimens if available. Quantification should neuroendocrine differentiation (MANEC), while a unique be performed at the “hot spot” within the tumor where staging protocol applies to low- and intermediate-grade mitotic activity is the highest. If the mitotic rate and pro- (G1 and G2) NETs. Since the current staging system is liferative index suggest different grades, the higher grade fairly new and remains controversial, it is important for is assigned. Of note, cytologic atypia in G1 tumors has no the pathologist to include all important elements in the effect on clinical behavior. G2 tumors may show punc- report, including the diagnosis, site of tumor/metastasis, tate necrosis, although necrosis is not considered a grading grade, stage, mitotic rate, proliferation rate, and results of criterion in abdominal NETs. immunohistochemical staining. Table 9.5 summarizes the The current grading and staging criteria for small differences between the staging criteria for small bowel bowel NETs are based on AJCC/UICC TNM, seventh NET and adenocarcinomas/NEC. Notably, if the size of edition. The cancer staging protocol for small bowel an NET exceeds 1 cm, the tumor is staged as pT2 even if adenocarcinoma is used for small bowel neuroendocrine it is confined to the submucosa and does not invade the carcinomas (G3) and tumors with mixed glandular/ muscularis propria. 2 3 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 9.5 Comparison of Staging Criteria for Small Bowel Adenocarcinoma and Small Bowel Neuroendocrine Tumor Small Bowel Adenocarcinoma and Poorly Small Bowel Well-Differentiated Neuroendocrine Differentiated Neuroendocrine Carcinoma Tumor pT1 Tumor invades lamina propria or submucosa Tumor invades lamina propria or submucosa and tumor size ≤1 cm pT2 Tumor invades muscularis propria Tumor invades muscularis propria or tumor size >1 cm pT3 Tumor invades through the muscularis propria into Tumor invades through the muscularis propria into the subserosa subserosal tissue without penetration of overlying or into the nonperitonealized perimuscular tissue serosa (jejunal or ileal tumors) (mesentery or retroperitoneum) with extension or invades pancreas or retroperitoneum (ampullary or ≤2 cm duodenal tumors) or into nonperitonealized tissues pT4 Tumor penetrates visceral peritoneum (serosa) Tumor penetrates visceral peritoneum (serosa) or or directly invades other organs or structures invades other organs (including other loops of small intestine, mesentery, or retroperitoneum >2 cm, and abdominal wall by way of serosa; for duodenum only, invasion of pancreas or bile duct) pN1 Metastases in one to three regional lymph nodes Metastases in regional lymph nodes pN2 Metastases in four or more regional lymph nodes − pM1 Distant metastasis Distant metastasis MESENCHYMAL TUMORS is variable. On a molecular level, KIT-activating muta- tions are common in small intestinal GISTs, and dupli- Mesenchymal tumors are discussed in detail in Chapter 5, cation of AY502-503 in KIT exon 9 is virtually specific thus the focus of this

chapter is on the mesenchymal neo- for small intestinal (vs. gastric) GISTs (see Chapter 5 for plasms that are most common in or unique to the small further discussion of immunohistochemical and molecu- bowel. lar aspects of GIST). Small intestinal GISTs are more likely to be malignant Gastrointestinal Stromal Tumors (GISTs) than gastric GISTS (40%–50% of small bowel tumors vs. 20%–25% of gastric GIST). The risk assessment for pri- GISTs are mesenchymal tumors that arise from the inter- mary GIST is based on tumor size, location, and mitotic stitial cells of Cajal. The small bowel is the second most activity. The major differential diagnosis includes other common location for GIST, preceded by the stomach spindle cell mesenchymal tumors, such as leiomyoma, (which accounts for 60% of GIST). The jejunum and schwannoma, and inflammatory fibroid polyp. This is ileum are the most commonly affected segments, followed also discussed in detail in Chapter 5. by duodenum. When compared with gastric GISTs, small intestinal GISTs (Figure 9.15A) are more likely to pres- Smooth Muscle Tumors ent with acute complications, such as intestinal obstruc- tion and tumor rupture. Histologically, small intestinal Leiomyomas are much less common in the small bowel than GISTs are typically spindle cell tumors (Figure 9.15B–C), in the esophagus or large intestine. These tumors can arise like their counterparts in other regions of the GI tract. either in association with the muscularis mucosae or with However, a few histologic features are specific to small the muscularis propria (Figure 9.16A). The cells are cytolog- bowel GISTs. Approximately half of small bowel GISTs ically bland, arranged in perpendicularly oriented fascicles, contain microscopically distinctive, extracellular collagen and have bright eosinophilic cytoplasm and blunt-ended globules known as “skeinoid fibers,” which are associ- nuclei (Figure 9.16B). Rare cases may show nuclear atypia, ated with a lower grade and favorable outcome (Figure but lack mitotic activity. Leiomyomas are immunoreactive 9.15D). In addition, nuclear palisading is less commonly with markers such as smooth muscle actin, smooth muscle seen in small intestinal tumors when compared to gas- myosin heavy chain (Figure 9.16C), myosin, desmin, and tric and colorectal GISTs. Focal nuclear pleomorphism caldesmon, but are typically negative for DOG1 and KIT. is more common in small intestinal GISTs from patients Leiomyosarcomas are extremely rare in the GI tract. They with NF1. usually arise in the muscularis propria, and show marked GISTs typically have strong and diffuse KIT and cytologic atypia, significant mitotic activity, and necrosis. DOG1 positivity (Figure 9.15E–F). Reactivity for other Similar to leiomyomas, leiomyosarcomas are actin and des- markers, including smooth muscle actin, S100, and CD34, min positive, and KIT negative. 9 Neoplasms of the Small Intestine 2 3 3 (A) (B) (C) (D) (E) (F) FIGURE 9.15 This gross photograph shows a large gastrointestinal stromal tumor (GIST) arising in the wall of the small bowel and bulging outward (A). The cut surface shows hemorrhage and degenerative changes. The cellular tumor is composed of fascicles of spindle cells (B–C). Skeinoid fibers can also be seen in small bowel GIST (D, arrows). GISTs diffusely express DOG1 (E) and KIT (F). 2 3 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 9.16 This gross photograph shows a leiomyoma arising from the wall of the small bowel (A). It has a tan–white, firm, whorled cut surface. At low power, the tumor is composed of cytologically bland spindle cells with elongated nuclei and eosinophilic cytoplasm, with low to medium cellularity (B). Leiomyomas show diffuse strong reactivity to smooth muscle myosin heavy chain (C). Fibroblastic Tumors fibroblastic and myofibroblastic proliferation that is associ- ated with CTNNB1 or APC mutations. Mesenteric fibro- Inflammatory fibroid polyps are benign polypoid submu- matosis usually expresses nuclear β-catenin (Figure 9.18C) cosal neoplasms; the ileum is the second most common site and estrogen receptor. Fibromatosis is associated with FAP in the GI tract, after the gastric antrum. These lesions can (Gardner syndrome). present as an obstructive mass and may cause intussuscep- tion. Histologically, inflammatory fibroid polyps are com- Lipomatous Lesions posed of loose fibromyxoid stroma containing spindle cells, inflammatory cells (particularly eosinophils, plasma cells, Lipomatous hypertrophy of the ileocecal valve is char- and mast cells), and prominent vessels that may have sur- acterized by a circumferential deposition of fat in the rounding concentric fibrosis (Figure 9.17A–D). The tumor submucosa of the ileocecal valve. Submucosal deposition cells are positive for CD34 and smooth muscle actin, and of small amounts of mature fat is common throughout negative for KIT and DOG1. the GI tract; however, the prominent fat deposition at Mesenteric fibromatosis (desmoid tumor) (Figure the ileocecal valve may result in an elevated mucosal 9.18A–B) originates in the mesentery, but may form a fibrotic protrusion into the lumen that produces a mass, result- mass involving the intestinal wall. This process is a clonal ing in occasional mucosal ulceration or even partial 9 Neoplasms of the Small Intestine 2 3 5 (A) (B) (C) (D) FIGURE 9.17 Inflammatory fibroid polyps are submucosal lesions composed of spindle cells within a loose fibromyxoid stroma (A–B). Higher power reveals cytologically bland spindle cells in a background of myxoid stroma with inflammatory cells including prominent eosinophils, lymphocytes, and plasma cells (C). Vessels are prominent, often surrounded by concentric collagen deposition (D). obstruction. The etiology of this process is unclear. Kaposi sarcoma may occur in the GI tract of HIV Lipomas can be seen in other locations in the small bowel patients with severe immunosuppression. The duodenum as well (Figure 9.19), but are more commonly located in is more commonly involved by Kaposi sarcoma than the the right colon. jejunum or ileum. Histologically, the tumor is composed of submucosal spindle cell proliferation, forming slit-like vas- Vascular Tumors cular channels containing red blood cells (Figure 9.20D–E). Extravasated red blood cells are frequently seen. The Hemangiomas and lymphangiomas are relatively common tumor cells are positive for CD31 (Figure 9.20F), HHV-8 mesenchymal lesions throughout the GI tract, and resem- (Figure 9.20G), CD34, and Factor VIII. Angiosarcoma of ble their counterparts elsewhere. Microscopically, multiple the small intestine is rare, and most often occurs in older cystically dilated spaces are lined by a single layer of flat- patients who were previously treated with radiation ther- tened endothelium. Hemangiomas generally contain blood, apy for uterine or urinary bladder cancer. The tumors are whereas lymphangiomas contain proteinaceous material composed of pleomorphic spindle cells or epithelioid cells (Figure 9.20A–B). In addition, lymphangiomas frequently that form primitive vascular channels (Figure 9.20H–I). contain lymphoid aggregates. Immunostains can be help- The tumor cells are positive for CD31, but show more ful in this distinction: D2-40 is more frequently positive in variable expression for CD34 or Factor VIII. One caveat is lymphangiomas (Figure 9.20C), whereas CD31 and CD34 that epithelioid variants may show keratin positivity, and are more frequently positive in hemangiomas. mimic a carcinoma. 2 3 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 9.18 Mesenteric fibromatosis (desmoid tumor) is composed of intersecting fascicles of bland spindle cells (A). The nuclei have smooth chromatin and inconspicuous nucleoli (B). There is diffuse nuclear expression of β-catenin (C). Gastrointestinal Clear Cell Sarcoma GI clear cell sarcoma is a rare tumor with a character- istic t(2;22)(q32;q12) chromosome translocation leading to EWSR1–CREB1 fusion. This tumor is more com- monly found in the small intestine than the stomach and colon. It often presents as a mural mass in young adults, with frequent metastases to mesenteric lymph nodes and liver. The tumor cells are rounded to mildly spindled (Figure 9.21A–B) and can contain multinucleated osteo- clast-like giant cells. The tumors share morphologic simi- larity with peripheral clear cell sarcoma and cutaneous malignant melanoma, and likewise they express S100. However, unlike the other two entities, GI clear cell sar- comas do not express HMB-45 and melan-A. A recent FIGURE 9.19 This lipoma of the small bowel consists ultrastructural examination demonstrates primitive neu- of a well-circumscribed submucosal mass composed of roectodermal differentiation in the tumor cells; there- mature adipocytes that are relatively uniform in size, and fore, a new designation of malignant GI neuroectodermal lack cytologic atypia. tumor has been proposed for this tumor type. 9 Neoplasms of the Small Intestine 2 37 (A) (B) (C) (D) (E) (F) FIGURE 9.20 This gross photo demonstrates a circumscribed, lobulated mucosal lesion containing milky white lymph, typical of lymphangioma (A). The lesion is centered in the submucosa, but the dilated, lymph-filled spaces extend into the mucosa (B). Multiple cystically dilated, endothelial-lined spaces are filled with thin pink amorphous material. D2-40 highlights the endothelial cells of the lymphatic channels (C). Kaposi sarcoma consists of a proliferation of spindle cells, forming slit-like vascular channels with red blood cells and extravasated red cells (D–E). (continued) 2 3 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (G) (H) (I) FIGURE 9.20 (continued) The tumor expresses CD31 (F) and HHV-8 (G). Angiosarcomas are composed of spindled or epithelioid cells that form primitive vascular channels (H–I). This example extends entirely through the wall of the small bowel. HEMATOLYMPHOID TUMORS also involve the small intestine. For additional informa- tion on a general approach to lymphoma diagnosis in the Overview GI tract, see Chapter 4. Any of the lymphomas described in this text can involve the small bowel, but several types have a propensity for Enteropathy-Associated T Cell Lymphoma occurring at this site, and thus will be discussed in detail (EATL) in this chapter. As in the rest of the GI tract, B cell lympho- This T cell lymphoma has historically been associated with mas are most common, but two unique T cell lymphomas underlying gluten-sensitive enteropathy (celiac disease), (A) (B) FIGURE 9.21 This gastrointestinal clear cell sarcoma from a 30-year-old woman shows sheets of epithelioid cells arranged in a vaguely nested pattern in the submucosa of the small bowel (A). The tumor cells show focal clearing of cytoplasm, small nucleoli, and scattered mitoses (B, courtesy of Dr. Paul Wakely Jr.). 9 Neoplasms of the Small Intestine 2 3 9 though more recently a subtype has been identified that perforation. This is due to the inherent characteristics of most often occurs outside the clinical setting, termed “Type the lymphoma and, often in the case of classic/Type I dis- II EATL.” A small number of patients who develop EATL ease, to the underlying effects of celiac disease. Median have a history of celiac disease diagnosed in childhood, survivals are measured in months. but more commonly the diagnosis occurs in adulthood or is made concurrently with the diagnosis of lymphoma. It occurs most commonly in the jejunum, where there is Extranodal NK/T Cell Lymphoma (ENKTL), overlap with what has in the past been termed “ulcerative Nasal Type jejunitis.” In this era of molecular clonality analysis, it has This relatively rare lymphoma is important to consider been demonstrated that patients with celiac disease who in the differential diagnosis of EATL in the small bowel. become refractory to a gluten-free diet, and subsequently While it is uncommon and usually found in the sinonasal progress to EATL, can harbor identical T cell clones in tract, as implied by its name, the most frequent extranasal their intestinal mucosa both before and after the develop- site of involvement by ENKTL is the GI tract. Like Type ment of lymphoma. In addition, the same clonal T cells can II EATL, the lymphoma cells of ENKTL usually express be found in other locations throughout the GI tract. This CD56, although they are usually negative for both CD4 finding suggests a spectrum of neoplastic disease in these and CD8. Surface CD3 is also negative, although cyto- patients. plasmic CD3ε can be found, depending on the specific EATL causes ulcerating masses that infiltrate transmu- anti-CD3 antibody used for immunohistochemistry (or rally (Figure 9.22A). This may lead to perforation, which flow cytometry). Cytotoxic molecules including TIA-1 may be the presenting sign of disease, even in patients who and granzyme B are also expressed. ENKTL is essen- have not been previously diagnosed with celiac disease. tially invariably related to Epstein–Barr virus (EBV), “Classical” (or Type I) EATL accounts for the majority which can be demonstrated by in situ hybridization EBV- of cases, and is usually associated with the celiac-disease- encoded ribonucleic acid (EBER) studies. related HLA DQ2 or DQ8 haplotypes. It consists of vari- The cells of ENKTL have variable morphology, rang- ably pleomorphic, intermediate- or large-sized cells with ing from

small lymphocytes to large and anaplastic cells. prominent nucleoli that most commonly express CD3 and When it involves mucosa, including that of the GI tract, CD7 (Figure 9.22B–C). They classically lose expression of ulceration is a frequent feature. An angiocentric and the pan-T cell marker CD5, and are CD4-negative. CD8 angiodestructive pattern of infiltration is quite character- is most often negative but may be expressed, and the neo- istic, with lymphoma cells overrunning and destroying the plastic cells have other evidence of a cytotoxic phenotype, walls of mural blood vessels, which can lead to extensive including expression of cytotoxic granule-associated pro- tissue necrosis (Figure 9.23). teins including TIA-1 and granzyme B. CD56 is negative and the cells express the αβ T cell receptor. Frequently, the Follicular Lymphoma (FL) neoplastic T cells express CD30, which may lead to confu- sion with other CD30-positive T cell lymphomas such as This mature B cell lymphoma occurs throughout the GI anaplastic large cell lymphoma (particularly when the cells tract, both as a primary tumor and as secondary involve- are markedly pleomorphic). Other morphologic signs of ment by a neoplasm centered elsewhere, such as in retro- celiac disease, from an isolated increase in intraepithelial peritoneal lymph nodes. It is discussed here because there lymphocytes to fully developed celiac-type changes with is also a primary form that appears to be unique to the mucosal flattening, may be present in the intact mucosa small intestine (often the duodenum), which, while mor- elsewhere in the intestine (Figure 9.22D). phologically and immunophenotypically indistinguishable In contrast, the monomorphic (Type II) variant is from other forms of FL, is believed to have a very good composed of monotonous, small- to intermediate-sized prognosis with conservative therapy. Nonetheless, it must cells with inconspicuous nucleoli and less cytoplasm be distinguished from systemic cases of FL by full staging (Figure 9.22E). The immunophenotype differs from the procedures including bone marrow biopsy. classic form, with most cases expressing CD8 (approxi- FL is composed of B lymphocytes with the charac- mately 80%) and CD56 (greater than 90%), along with teristic features of follicle center (or germinal center) CD3 and CD7. CD5 is negative, and this variant may cells, and most often has a nodular or follicular pattern express either the αβ or γδ T cell receptor. These differ- (Figure 9.24A). The neoplastic cells have variable mor- ences, along with the lack of close association with celiac phology, some with small, cleaved nuclei and scant cyto- disease and the HLA DQ2 and DQ8 haplotypes, have plasm characteristic of follicular “centrocytes,” and others led some to question whether this disease should truly be that are large with open chromatin and more abundant regarded as a variant of EATL or as a separate disease. cytoplasm characteristic of “centroblasts” (Figure 9.24B). In either form, EATL has a very aggressive course and FL is graded, based on the proportion of centroblast-like a poor prognosis, particularly in the setting of intestinal cells, into three grades that have prognostic significance. 2 4 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 9.22 Enteropathy-associated T cell lymphoma (EATL) arising in the small intestine and infiltrating transmurally (A). The lymphoma involves the mucosa, penetrates the fibers of the muscularis propria (arrow), and involves the subserosa. The cells of classical EATL are intermediate to large, with pleomorphic nuclei and occasional prominent nucleoli (B). Endothelial cells of admixed blood vessels (arrow) are a convenient internal standard for large nuclear size. Note the presence of scattered eosinophils (arrowhead). Immunohistochemically (C), most cases are positive for the pan-T cell markers CD3 (left panel) and CD7 (center panel). CD5 (right panel) is usually negative. The positive-staining cells are scattered non-neoplastic T cells. A careful examination of the mucosa of uninvolved small intestine will very often reveal the presence of sprue-type changes indicating underlying celiac disease, particularly in classic (non-Type II) EATL. In this panel (D), the mucosa is flattened, with numerous intraepithelial lymphocytes on the surface and crypt epithelium. There is also a prominent lamina propria lymphoplasmacytic infiltrate. Studies have shown that the intraepithelial lymphocytes even in areas away from the lymphoma can be clonal. The so-called Type II EATL (E) is less often associated with underlying celiac disease and tends to be composed of monotonous, small- or intermediate-sized cells (left panel) that often express CD56 (right panel). Note the relatively intact villous architecture of the overlying mucosa in the right panel. 9 Neoplasms of the Small Intestine 2 41 CD10 and/or BCL-6 positive that also expresses BCL-2 is diagnostic of FL. The unique “primary intestinal” FL mentioned earlier is morphologically identical to other forms, but is local- ized to the small intestine, very often the duodenum. It is more common in young or middle-aged patients, often women, and can appear as a polyp or polyps endoscopi- cally (Figure 9.24E). There is evidence that patients with this form of the disease have a very good prognosis with endoscopic “excision” of the polyp alone. The only way to be certain of the primary intestinal form of the disease, however, is to perform full clinical staging to rule out sys- temic involvement. Systemic FL is relentlessly progressive and incurable, with a significantly worse prognosis than the localized small intestinal form. FIGURE 9.23 Extranodal NK/T cell lymphoma, nasal type. This rare lymphoma occurs in the head and Burkitt Lymphoma (BL) neck, but the GI tract is the most commonly involved BL occurs in endemic and sporadic forms, as well as in extranasal site. Histologically, it bears a resemblance to an immunodeficiency-related variety. The classic de novo EATL (especially the classic type), with relatively large presentation of the sporadic form of this rare B cell lym- and pleomorphic cells. It is essentially always related phoma is in the ileocecal region, where it can create large to underlying Epstein–Barr virus and has a distinctive, masses that grow at a seemingly impossible rate. In the angiocentric and angiodestructive infiltration pattern. Here, lymphoma cells invade and disrupt the wall of a area of the ileocecal valve, this can lead to dramatic clini- medium-sized vessel (arrow). cal presentations that include rapid-onset obstruction and perforation (Figure 9.25A). Most patients are relatively young, although immunodeficiency-associated cases, many of which are related to underlying HIV infection, Grade 3 is further subdivided into grades 3A and 3B (see occur in older patients. EBV infection is a fairly frequent Table 9.6). Current practice is to combine grades 1 and association with BL, especially in the endemic form, but 2 into an effective “low-grade” category, reported as also in sporadic and immunodeficiency-associated cases. “follicular lymphoma, grade 1–2.” As with other B cell The endemic form is limited to equatorial Africa and New lymphomas, the appearance of a significant (reported as Guinea and will not be discussed further. greater than 25% by some authorities) diffuse large cell The cells of BL are intermediate in size with round, component should be regarded and reported as diffuse monotonous nuclei and one or more relatively inconspicu- large B cell lymphoma (DLBCL) with a precursor FL in ous nucleoli (Figure 9.25B). In touch or aspirate prepara- the background (Figure 9.24C). tions, perinuclear cytoplasmic vacuoles may be seen, but The characteristic nodular/follicular pattern, along these are not usually apparent in formalin-fixed tissue with the monotonous appearance of the lymphoma cells preparations. A characteristic “starry sky” pattern reflects making up these nodules, makes it possible to essentially the extremely rapid proliferative rate, with numerous tingi- render a definite diagnosis even on routine H&E stained ble-body macrophages interspersed with the monotonous sections in classic cases. Nonetheless, a typical immu- lymphoma cells (Figure 9.25C). This proliferative rate can nohistochemical panel shows that the lymphoma cells be confirmed using Ki-67 immunohistochemistry, which express pan-B cell markers including CD20 and CD79a, will reveal near-100% labeling in the lymphoma cells as well as the markers of follicle center cell differentiation (Figure 9.25D). The neoplastic cells are CD20-positive B CD10 and BCL-6. As noted in Chapter 4, the diagnosis cells and have a germinal center phenotype, expressing of FL and, in particular, its separation from a reactive fol- BCL-6 and CD10. Unlike many other B cell lymphomas, BL licular hyperplasia that can accompany many conditions, is usually BCL-2 negative, a finding that helps distinguish is greatly aided by the use of a BCL-2 immunohistochemi- it from the diffuse form of FL. The diagnosis can be con- cal stain (Figure 9.24D). While many B cell lymphomas, firmed with molecular studies for translocations involving along with numerous normal hematolymphoid cells, the MYC gene on chromosome 8, which are almost invari- express this protein, it is negative in reactive, non-neo- ably present. By far, the most common of these is t(8;14), plastic germinal centers. In contrast, the vast majority of which brings together MYC and the IGH gene, but t(2;8) FL, including nearly all low-grade (grades 1–2) tumors, and t(8;22) are also found, involving the genes for kappa are BCL-2 positive. Thus, a follicular proliferation that is and lambda light chains, respectively. 2 42 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 9.24 Follicular lymphoma involving the small intestine. Here, a nodular lymphoid aggregate with a follicular architecture expands the enteric mucosa (A). At high magnification (B), the infiltrate of follicular lymphoma contains a mixture of small cells with irregular nuclear contours (centrocytes) and larger cells with more vesicular nuclei and prominent nucleoli (centroblasts; arrow). The centrocytes often have a nuclear indentation or cleft (“cleaved” nuclei; arrowhead), which is best seen by changing the plane of focus slightly under the microscope. This low-grade case has a preponderance of centrocytes. Cases with sheets of large and pleomorphic cells that do not have a follicular pattern, such as this case (C), should be diagnosed as diffuse large B cell lymphoma (DLBCL). If other areas of the lymphoma have a recognizable precursor follicular lymphoma, this should be mentioned in the diagnosis, as the DLBCL is potentially curable, but the underlying follicular lymphoma may be relatively resistant to therapy. The neoplastic follicles in follicular lymphoma (D) are CD10 positive (left panel), indicating their follicle center cell phenotype. BCL-6 is also positive. Note the CD10 staining of the brush border on the luminal side of the epithelium as well. In addition, BCL-2 is positive in the neoplastic follicles (right panel), a useful feature in distinguishing follicular lymphoma (positive) from reactive follicular hyperplasia (negative). This duodenal follicular lymphoma (E) had a polypoid endoscopic appearance, which is reflected in the low- power histological appearance as well. In this case, the lymphoma was found only in the small intestine, making it a case of so-called “primary intestinal” follicular lymphoma. 9 Neoplasms of the Small Intestine 2 4 3 TABLE 9.6 Grading Scheme for Follicular Lymphoma As introduced in Chapter 4 and further discussed in Chapter 8, in association with DLBCL, a recently recog- Lymphoma nized subset of B cell lymphoma has been found to harbor Grade Features genetic abnormalities MYC, as well as BCL2 and/or BCL6. These so-called “double-hit” lymphomas are currently 1 0–5 centroblasts/HPF Low grade 2 6–15 centroblasts/HPF included in the nebulous category of “B cell lymphoma, 3A >15 centroblasts/HPF with centrocytes High grade unclassifiable, with features intermediate between dif- 3B >15 centroblasts/HPF without fuse large B cell lymphoma and Burkitt lymphoma” in the centrocytes (ie, all centroblasts in most recent (2008) WHO classification. These lymphomas follicular structures) may be categorized differently in subsequent WHO clas- Abbreviation: HPF = high-power field (40x objective). Counts are made by sifications but, for now, it is thought that their recognition counting 10 HPF (when available) and dividing by 10. and separation from BL and DLBCL has prognostic, and (A) (B) (C) (D) FIGURE 9.25 Burkitt lymphoma can cause clinically impressive changes, including large masses that grow at a seemingly impossible rate, and which can result in perforation (arrows) as in this case (A). Histologically, Burkitt lymphoma is composed of intermediate-sized lymphocytes that are quite monomorphic (B), with fine nuclear chromatin and, often, several inconspicuous nucleoli (arrows). The lymphoma has a brisk proliferative rate, as evidenced by several mitotic figures in this single field (arrowheads). The rapid proliferation of Burkitt lymphoma is also reflected in the so-called “starry sky” appearance at low magnification (C). The lighter colored “stars” in the monotonous field of lymphoma cells are tingible-body macrophages, which contain nuclear

and cellular debris. The final confirmation of Burkitt lymphoma’s impressive proliferative ability is a Ki-67 immunostain (D), which usually reveals a proliferative rate (as indicated by positive nuclear staining) of nearly 100%. 2 4 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide potentially therapeutic, significance, especially in younger Metastatic tumors typically present as intestinal wall patients. Double-hit lymphoma can strongly resemble BL, thickening with submucosal spread, but may present as with a high proliferative rate (though usually not 100%) mucosal polyps or nodules (Figure 9.26A–C). In the lat- and a germinal center cell immunophenotype. It may be ter situation, it can be challenging to distinguish meta- either BCL-2 positive or negative, and molecular assays for static from primary carcinomas, due to both morphologic the genetic abnormalities described earlier in this paragraph overlap and to mucosal spread of some metastatic tumors, are the only way to confirm the diagnosis at this point. which mimics in situ growth and suggests an adenoma- BL is a very clinically aggressive neoplasm. While this tous precursor lesion. Because of this propensity for muco- behavior leads to impressive and life-threatening clinical sal spread, the finding of an apparent precursor lesion and presentations, it also means that BL is potentially curable growth along a basement membrane cannot be reliably when subjected to appropriate therapy. Depending on patient used to distinguish primary from metastatic lesions in age, long-term survival rates range from 70% to 90% when the small bowel. This is especially problematic in cases of intensive chemotherapy is employed. metastases from other sites in the GI or pancreaticobiliary tract, (Figure 9.27A–B) or gynecologic adenocarcinomas. In most cases, the history and radiographic findings METASTASES TO THE SMALL BOWEL are sufficient to determine the site of origin in potentially metastatic cases. It can be extremely helpful to review the Metastases to the small intestine are 2.5 times more primary tumor if it is available for comparison. In dif- common than primary small bowel carcinomas in large ficult cases, immunohistochemical stains can be used to autopsy studies. The most common metastatic tumors to help determine the site of origin. An initial panel of CK7, the small intestine include tumors from the skin (particu- CK20, and CDX2 is helpful in ascertaining the site of larly melanoma), breast, ovary, lung, and pancreas. origin. Additional commonly used markers include TTF1 (A) (B) (C) FIGURE 9.26 This example of metastatic melanoma to the small bowel presented as a mucosal polyp, mimicking a primary tumor (A). Another example of metastatic melanoma to the small bowel demonstrates a growth pattern centered in the submucosa, which extends into the surface epithelium (B). The tumor cells are dyscohesive with a plasmacytoid appearance and prominent nucleoli (C), typical of melanoma. 9 Neoplasms of the Small Intestine 2 4 5 (A) (B) FIGURE 9.27 Intramucosal involvement of small intestine by a metastatic adenocarcinoma of the appendix (A). The metastatic tumor glands inconspicuously merge with the small intestinal epithelium at the surface (B) mimicking the in situ component of a small intestinal primary. (lung, thyroid), ER/PR (breast, Müllerian), PAX8 (kidney, Schottenfeld D, Beebe-Dimmer JL, Vigneau FD. The epidemiology and Müllerian, thyroid), p53 and WT1 (serous carcinoma with pathogenesis of neoplasia in the small intestine. Ann Epidemiol. the latter also expressed by mesothelioma), napsin A (lung 2009;19:58–69. Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. or papillary renal cell carcinoma), and prostate-specific 2013;63:11–30. antigen (prostate). Attention to the extent and intensity of Umar A, Boland R, Terdiman JP, et al. Revised Bethesda guidelines for the immunoreactivity is sometimes helpful in appreciating hereditary nonpolyposis colorectal cancer (Lynch syndrome) and the subtle differences in those tumors with morphologic microsatellite instability. J Nat Cancer Inst. 2004;96:261–268. van Ree K, Thurley P, Singh R, et al. The imaging features of small and frequent immunophenotypic similarities. As always, bowel tumours. J Gastrointest Cancer. 2012;43:405–412. immunohistochemical stains are most useful when used in panels with attention to the clinical findings. Reactive/Benign Lesions Chen ZM, Scudiere JR, Abraham SC, et al. Pyloric gland adenoma: an entity distinct from gastric foveolar type adenoma. Am J Surg SELECTED REFERENCES Pathol. 2009; 33:186–193. Genta RM, Kinsey RS, Singhal A, et al. Gastric foveolar metaplasia and General gastric heterotopia in the duodenum: no evidence of an etiologic role for Helicobacter pylori. Hum Pathol. 2012;41:1593–1600 Ashley SW, Wells SA. Tumors of the small intestine. Semin Oncol Lambert MP, Heller DS, Bethel C. Extensive gastric heterotopia of 1988;15:116–128. the small intestine resulting in massive gastrointestinal bleeding, Bellizzi AM. Immunohistochemistry in gastroenterohepatopancrea- bowel perforation, and death: report of a case and review of the tobiliary epithelial neoplasia: Practical application, pitfalls, and literature. Pedatr Dev Pathol. 2000;3:277–280. emerging markers. Surg Pathol Clin. 2013;6:567–609. Limaiem F, Haddad I, Marsaoui L, et al. Pancreatic heterotopia of the Bosman F, Carneiro F, Hruban R, et al (Eds.). Tumours of the small small intestine: two case reports. Pathologica. 2013;105:18–20. intestine. In World Health Organization Classification of Uppal K, Tubbs RS, Matusz P, et al. Meckel’s diverticulum: a review. Tumours of the Digestive System. 4th ed. Lyon, France: IARC Clin Anat. 2011;24:416–422. Press; 2010:chap 6. Darling RC and Welch CE. Tumors of the small intestine. N Engl J Med. 1959;260:397–408. Hamartomatous Polyp Syndromes Dye CE, Gaffney RR, Dykes TM, et al. Endoscopic and radiographic evaluation of the small bowel in 2012. Am J Med. 2012;125:1228. Adolph VR and Bernabe K. Polyps in Children. Clin Colon Rectal Surg. e1–1228.e12 2008;21:280–285. Piscaglia AC, Campanale M, Gasbarrini G. Small bowel nonendocrine Arber N and Moshkowitz M. Small bowel polyposis syndromes. Curr neoplasms: current concepts and novel perspectives. Eur Rev Med Gastroenterol Rep. 2011;13:435–441. Pharmacol Sci. 2010;14:320–326. McGarrity TJ and Amos C. Peutz-Jeghers syndrome: clinicopath- Rampertab SD, Forde KA, Green PH. Small bowel neoplasia in coeliac ology and molecular alterations. Cell Mol Life Sci. 2006;63: disease. Gut. 2003;52:1211–1214. 2135–2144. 2 4 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Adenomatous Polyps Planck M, Ericson K, Piotrowska Z, et al. Microsatellite instability and expression of MLH1 and MSH2 in carcinomas of the small intes- Genta RM, Feagins LA. Advanced precancerous lesions in the tine. Cancer. 2003;97:1551–1557. small bowel mucosa. Best Pract Res Clin Gastroenterol. Potter DD, Murray JA, Donohue JH, et al. The role of defective mis- 2013;27:225–233. match repair in small bowel adenocarcinoma in celiac disease. Jass JR. Colorectal polyposes: from phenotype to diagnosis. Pathol Res Cancer Res. 2004;64:7073–7077. Pract. 2008;204:431–447. Raghav K, Overman MJ. Small bowel adenocarcinomas—exist- Kadmon M, Tandara A, Herfarth C. Duodenal adenomatosis in famil- ing evidence and evolving paradigms. Nat Rev Clin Oncol. ial adenomatous polyposis coli. A review of the literature and 2013;10:534–544. results from the Heidelberg Polyposis Register. Int J Colorectal Richir M, Songun I, Wientjes C, et al. Small Bowel Adenocarcinoma Dis. 2001; 16:63–75 in a Patient with Coeliac Disease: Case Report and Review of the Koornstra JJ. Small bowel endoscopy in familial adenomatous pol- Literature. Case Rep Gastroenterol. 2010;4:416–420. yposis and Lynch syndrome. Best Pract Res Clin Gastroenterol. Zouhairi ME, Venner A, Charabaty A, et al. Small bowel adenocarci- 2012;26:359–368. noma. Curr Treat Options Oncol. 2008;9:388–399. Martin JA and Haber GB. Ampullary adenoma: clinical manifesta- tions, diagnosis, and treatment. Gastrointest Endosc Clin N Am. 2003;13:649–669 Neuroendocrine Tumors Klimstra DS. Pathology reporting of neuroendocrine tumors: essential elements for accurate diagnosis, classification, and staging. Semin Adenocarcinoma Oncol. 2013;40:23–36. Benhammane H, El M’rabet FZ, Idrissi Serhouchni K, et al. Small Maggard MA, O’Connell JB, Ko CY. Updated population-based review bowel adenocarcinoma complicating coeliac disease: a report of carcinoid tumors. Ann Surg. 2004;240:117–122. of three cases and the literature review. Case Rep Oncol Med. Soga J, Tazawa K. Pathologic analysis of carcinoids. Histologic reevalu- 2012;2012:935183. ation of 62 cases. Cancer. 1971;28:990–998. Bilimoria KY, Bentrem DJ, Wayne JD, et al. Small bowel cancer in the Soga J. Carcinoids of the small intestine: a statistical evaluation of United States: changes in epidemiology, treatment, and survival 1102 cases collected from the literature. J Exp Clin Cancer Res. over the last 20 years. Ann Surg. 2009;249:63–71. 1997;16:353–363 Canavan C, Abrams KR, Mayberry J. Meta-analysis: colorectal and Strosberg J. Neuroendocrine tumours of the small intestine. Best Pract small bowel cancer risk in patients with Crohn’s disease. Aliment Res Clin Gastroenterol. 2012;26:755–773. Pharmacol Ther. 2006;23:1097–1104. Yang Z, Tang LH, Klimstra DS. Gastroenteropancreatic neuroendo- Chang HK, Yu E, Kim J, et al. Adenocarcinoma of the small intestine: crine neoplasms: historical context and current issues. Semin a multi-institutional study of 197 surgically resected cases. Hum Diagn Pathol. 2013;30:186–196. Pathol. 2010;41:1087–1096. Yantiss RK, Odze RD, Farraye FA, et al. Solitary versus multiple car- Diosdado B, Buffart TE, Watkins R, et al. High-resolution array cinoid tumors of the ileum: a clinical and pathologic review of 69 comparative genomic hybridization in sporadic and celiac dis- cases. Am J Surg Pathol. 2003;27:811–817. ease-related small bowel adenocarcinomas. Clin Cancer Res. Yao JC, Hassan M, Phan A, et al. One hundred years after “carci- 2010;16:1391–401. noid”: epidemiology of and prognostic factors for neuroendo- Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A eds. crine tumors in 35,825 cases in the United States. J Clin Oncol. AJCC cancer staging manual. New York, NY: Springer; 2010. 2008;26:3063–3072. Fu T, Pappou EP, Guzzetta AA, et al. CpG island methylator phenotype- positive tumors in the absence of MLH1 methylation constitute a Mesenchymal Tumors distinct subset of duodenal adenocarcinomas and are associated with poor prognosis. Clin Cancer Res. 2012;18:4743–4752. Akbulut S. Intussusception due to inflammatory fibroid polyp: a case Goodman MT, Matsuno RK, Shvetsov YB. Racial and ethnic varia- report and comprehensive literature review. World J Gastroenterol. tion in the incidence of small-bowel cancer subtypes in the United 2012; 18:5745–5752 States, 1995-2008. Dis Colon Rectum. 2013;56:441–448. Al Ali J, Ko HH, Owen D, et al. Epithelioid angiosarcoma of the small Green PH, Rampertab SD. Small bowel carcinoma and coeliac disease. bowel. Gastrointest Endosc 2006; 64:1018–1021 Gut. 2004; 53:774. Arora M, Goldberg EM. Kaposi sarcoma involving the gastrointestinal Haan JC, Buffart TE, Eijk PP, et al. Small bowel adenocarcinoma copy tract. Gastroenterol Hepatol (NY) 2010; 6:459–462 number profiles are more closely related to colorectal than to gas- Boyle L, Lack EE. Solitary cavernous hemangioma of small intestine. tric cancers. Ann Oncol. 2012;23:367–374. Case report and literature review. Arch Pathol Lab Med. 1993; Neely D, Ong J, Patterson J, et al. Small intestinal adenocarcinoma: 117:939–941 rarely considered, often missed? Postgrad Med J. 2013;89: Cabaud PG and Harris LT. Lipomatosis of the Ileocecal Valve. Ann 197–201. Surg. 1959;150:1092–1098. Nilubol N, Scherl E, Bub DS, et al. Mucosal dysplasia in ileal pelvic Chang CW, Wang TE, Chang WH, et al. Unusual presentation of pouches after restorative proctocolectomy. Dis Colon Rectum. desmoid tumor in the small intestine: a case report. Med Oncol. 2007;50:825–831. 2011;28:159–162 Overman MJ, Hu CY, Kopetz S, et al. A population-based comparison Chatterjee C, Khan D, U De. A rare presentation of gastrointestinal of adenocarcinoma of the large and small intestine: insights into a stromal tumors as a small bowel perforation: A single institution rare disease. Ann Surg Oncol. 2012;19:1439–1445. based clinical experience of three cases. J Dr. NTR Univ. Health Overman MJ, Hu CY, Wolff RA, et al. Prognostic value of lymph Sci. 2013;2:118–121 node evaluation in small bowel adenocarcinoma: analysis of the Corless CL. Gastrointestinal stromal tumors: what do we know now? surveillance, epidemiology, and end results database. Cancer. Mod Pathol. 2014;Suppl 1: S1–S16 2010;116:5374–5382. Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on Overman MJ. Rare but real. Management of Small Bowel morphology, molecular pathology, prognosis, and differential Adenocarcinoma. Am Soc Clin Oncol Educ Book. 2013:189–193. diagnosis. Arch Pathol Lab Med. 2006;130:1466–1478. Pan SY and Morrison H. Epidemiology of cancer of the small intestine. Morris-Stiff G, Falk GA, EL-Hayek K, et al. Jejunal cavernous lymph- World J Gastrointest Oncol. 2011;3:33–42. angioma. BJM Case Rep. 2011; pii:bcr0320114022 9 Neoplasms of the Small Intestine 2 47 Ni Q, Shang D, Peng H, et al. Primary angiosarcoma of the small intes- Hawkes EA, Wotherspoon A, Cunningham D. Diagnosis and man- tine with metastasis to the liver: a case report and review of the agement of rare gastrointestinal lymphomas. Leuk Lymphoma. literature. World J Surg Oncol. 2013; 11:242 2012;53:2341–2350. Pahwa M, Girotra M, Rautela A, et al. Periampullary leiomyosarcoma O’Malley DP, Goldstein NS, Banks PM. The recognition and classifi- presenting with cutaneous metastases: a rare entity. South Med J. cation of lymphoproliferative disorders of the gut. Hum

Pathol. 2010;103:1190–1191 2014;45:899–916. Rampone B, Pedrazzani C, Marrelli D, et al. Updates on abdominal Smith LB, Owens SR. Gastrointestinal lymphomas: entities and mimics. desmoid tumors. World J Gastroenterol. 2007;13:5985–5988 Arch Pathol Lab Med. 2012;136:865–870. Regula J, Wronska E, Pachlewski J. Vascular lesions of the gastro- Swerdlow SH, Jaffe ES, Brousset P, et al. Cytotoxic T-cell and NK-cell intestinal tract. Best Pract Res Clin Gastroenterol. 2008; 22: lymphomas: current questions and controversies. Am J Surg 313–328 Pathol. 2014;38:e60–e71. Stockman DL, Miettinen M, Suster S, et al. Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reap- Metastases praisal of clear cell sarcoma-like tumors of the gastrointestinal Disibio G, French SW. Metastatic patterns of cancers: results from a tract. Am J Surg Pathol. 2012;36:857–868 large autopsy study. Arch Pathol Lab Med. 2008;132:931–939. Suryawanshi KH, Patil TB, Damle RP, et al. Gastrointestinal stromal Estrella JS, Wu TT, Rashid A, et al. Mucosal colonization by metastatic tumour of small intestine presenting as a mesenteric mass. J Clin carcinoma in the gastrointestinal tract: a potential mimic of pri- Diagn Res. 2014;8:FD14–FD16 mary neoplasia. Am J Surg Pathol. 2011;35:563–572. Wysocki AP, Taylor G, Windsor JA. Inflammatory fibroid polyps Hess KR, Varadhachary GR, Taylor SH, et al. Metastatic patterns in of the duodenum: a review of the literature. Dig Surg. 2007; adenocarcinoma. Cancer. 2006;106:1624–1633. 24:162–168 Lianos GD, Messinis T, Doumos R, et al. A patient presenting with acute abdomen due to metastatic small bowel melanoma: a case Hematolymphoid Neoplasms report. J Med Case Rep. 2013; 7:216. Newton RC, Penney N, Nind N, et al. Small bowel malignant mela- Burke JS. Lymphoproliferative disorders of the gastrointestinal tract: a noma presenting as a perforated jejunal diverticulum: a case report review and pragmatic guide to diagnosis. Arch Pathol Lab Med. and literature review. Gastroenterol Rep (Oxf). 2014; pii:gou058. 2011;135:1283–1297. [Epub ahead of print] Damaj G, Verkarre V, Delmer A, et al. Primary follicular lymphoma Sundersingh S, Majhi U, Chandrasekar SK, et al. Metastatic malignant of the gastrointestinal tract: a study of 25 cases and a literature melanoma of the small bowel-report of two cases. J Gastrointest review. Ann Oncol. 2003;14:623–629. Cancer. 2012;43:332–335. 10 Neoplasms of the Appendix RHONDA K. YANTISS INTRODUCTION Enterochromaffi n (EC) Cell Neuroendocrine Tumors Most appendiceal neoplasms are incidentally detected in appendectomy or colectomy specimens obtained for other As mentioned in Chapter 3, the majority of appendi- indications. Slightly more than 50% of tumors are neuro- ceal NETs are composed of serotonin-producing entero- endocrine tumors (NETs), most of which share biochemical chromaffin (EC) cells, similar to jejunoileal tumors. properties with similar lesions of the distal small intes- Previously, these tumors have been known as “classic” tine. The majority of the remaining non-n euroendocrine carcinoid tumors. These tumors show a predilection for appendiceal tumors are mucinous in nature, whereas non- the distal appendiceal tip, where they form solitary yel- mucinous neoplasms, including serrated lesions, villous low nodules (Figure 10.1A–B). They are unencapsulated, adenomas, and nonmucinous adenocarcinomas, are rela- well-circumscribed aggregates of tightly packed nests tively less common. and acini (Figure 10.2A–B), the latter of which harbor Mucinous neoplasms are usually limited to the mucosa luminal material that is positive for periodic acid–Schiff (ie, adenomas), but they may invade the appendiceal wall stain, but do not contain mucin. Tumor cells contain or disseminate in the peritoneal cavity, resulting in sub- abundant faintly eosinophilic or clear cytoplasm and are stantial disease-related morbidity and mortality despite the both argentaffin and argyrophil positive. Some tumor relatively bland appearance of neoplastic epithelium. The cells contain brightly eosinophilic cytoplasmic granules. discordance between the histologic features and biologic The nuclei are typically round with a stippled chroma- behavior among mucinous tumors, and the infrequent tin pattern likened to “salt and pepper.” Nucleoli tend nature of nonmucinous lesions, has generated considerable to be small, but conspicuous, and mitotic figures are confusion regarding their nomenclature and classification. infrequent. Occasional tumors display degenerative-type nuclear atypia with hyperchromasia and multinucleation, which has no bearing on their biologic behavior. EC cells NEUROENDOCRINE TUMORS show strong immunopositivity for chromogranin A, syn- OF THE APPENDIX aptophysin, and serotonin. Most (70%) of these NETs are smaller than 1 cm in diameter and show a very low Ki-67 Overview proliferation index (less than 1%). Neuroendocrine tumors (NETs) are identified in less than 1% of patients undergoing appendectomy, and are more Enteroglucagon (L) Cell Endocrine Tumors common among adults. Patients tend to be slightly younger Approximately 10% to 20% of appendiceal NETs are com- than those with other NETs of the gastrointestinal tract, posed of enteroglucagon (L) cells. These tumors are small and lesions show a slight female predominance. It is likely (2–3 mm) nodules composed of trabeculae and cords of that these features reflect the frequency of appendectomy cytologically bland tumor cells. Some tumors display a pre- procedures among young patients and women undergoing dominantly acinar, or tubular, growth pattern with inspis- gynecologic surgery. sated mucin; hence their previous classification as tubular 2 4 9 2 5 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 10.1 This appendiceal neuroendocrine (carcinoid) tumor forms a yellow nodule in the distal appendix. The tumor obliterates the muscularis propria (arrow) (A, courtesy of Dr. Henry Appelman). This appendix cut in cross section illustrates the firm, yellow appearance of the carcinoid tumor on the cut section. The tumor infiltrates the mesoappendix (B, courtesy of Dr. George Gray). NETs or tubular carcinoids (Figure 10.2C–D). Lesions with aggressively first spread to regional lymph nodes followed either a trabecular or tubular appearance harbor enteroglu- by the liver, similar to NETs of other organs in the gas- cagons, glucagon-like peptides, pancreatic polypeptide, and trointestinal tract. Tumors that metastasize to the liver or peptide YY. Small red basophilic neuroendocrine granules produce bulky peritoneal disease may produce the carci- may be visible. The tumor cells are argyrophilic and show noid syndrome. Management decisions are largely based immunopositivity for chromogranin B, glucagon, and car- on tumor size. Small lesions (up to 1 cm in diameter) are cinoembryonic antigen (CEA) but do not stain for chro- adequately treated with appendectomy alone, provided the mogranin A. The Ki-67 proliferation index is usually very resection margin is negative. Right colectomy with lymph low. Because of the infiltrative growth pattern and the lack node dissection is generally limited to tumors larger than of chromogranin A expression, tubular carcinoids may be 2 cm or those with vascular invasion since these tumors mistaken for metastatic carcinoma. have a greater likelihood of regional lymph node involve- ment. Management of tumors ranging from 1 to 2 cm in Pathologic Reporting of Appendiceal diameter is somewhat controversial. Completely resected Endocrine Tumors tumors may be treated with appendectomy and clinical follow-up. Features suggestive of aggressive behavior (eg, Appendiceal neuroendocrine neoplasms are now included invasion of the mesoappendix, vascular invasion, mitotic in the American Joint Committee on Cancer Staging activity, and high Ki-67 labeling) may prompt right col- Manual and, thus, should be graded and staged in accor- ectomy and lymph node dissection, although the added dance with the Tumor Nodes Metastases (TNM) staging benefit of extensive surgery in these patients has not been system. Grading is determined based on a combination of clearly documented in the literature. mitotic activity and immunolabeling with Ki-67, as dis- cussed in Chapter 3, and thus Ki-67 staining should be per- formed in all cases. Other important criteria, such as the MUCINOUS NEOPLASMS OF THE APPENDIX status of the resection margin, presence of lymphovascular AND PERITONEUM invasion, and involvement of mesoappendiceal soft tissues, should also be included in surgical pathology reports. Overview The low-grade features of appendiceal mucinous neo- Biologic Behavior and Management plasms and confusing, descriptive terminology used to Appendiceal NETs usually behave in a benign fashion, classify them have led to several misconceptions regard- especially if they are small. Indeed, less than 1% of tumors ing their biologic potential. Older reports described spanning less than 2 cm metastasize. Those that do behave appendiceal adenomas that metastasized to the abdomen, 10 Neoplasms of the Appendix 2 51 (A) (B) (C) (D) FIGURE 10.2 This classic neuroendocrine tumor forms a nodule in the distal appendix, obliterating the lumen and expanding the submucosa. The tumor consists of cellular nests enmeshed in collagenous stroma (A). Lesional cells contain abundant, faintly eosinophilic cytoplasm and round nuclei with stippled chromatin and small nuclei (B). In contrast to classic tumors, hindgut neuroendocrine tumors contain cords and clusters of cells in a fibroblast-rich, cellular stroma (C). Some of these lesions contain small acinar structures and tubules. Lesional cells are polarized around a central lumen. The nuclei have smooth contours and small nucleoli (D). producing clinical findings of pseudomyxoma peritonei, tumors that have not yet seeded the peritoneum, but which resulted in their historical classification as tumors extend into the appendiceal wall or show mucin on the of uncertain malignant potential or adenomucinosis. serosal surface, remain poorly defined and somewhat However, it is now clear that mucinous tumors confined controversial. to the appendiceal mucosa are generally benign with no biologic risk, whereas those with peritoneal mucin Mucinous Adenoma and deposits pursue an indolent but relentless course and thus Mucinous Cystadenoma many authors classify these tumors as well- differentiated mucinous carcinomas for treatment and staging pur- Some appendiceal adenomas do not produce any gross poses. Criteria for classification of appendiceal mucinous abnormalities, whereas others cause localized or fusiform 2 5 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide appendiceal dilatation (Figure 10.3A–C). Although these denote lesions confined to the mucosa that show neoplas- lesions have been termed mucinous cystadenomas in the tic mucinous epithelium with a villous, undulating, or flat past, they are now mostly considered to be adenomas. By appearance at the luminal surface (Figure 10.5A). Others, definition, mucinous adenomas are confined to the appen- including the World Health Organization, use the term diceal mucosa and, with rare exception, do not display “low-grade appendiceal mucinous neoplasm” to describe mucin on the serosal surface (Figure 10.3D). Some authors cases that show mucin and/or epithelium that shows a require the presence of intact muscularis mucosae to make broad “pushing” front, rather than an infiltrative pattern a diagnosis of adenoma, and consider atrophy and fibro- of invasion (Figure 10.5B–D), as well as those that display sis of the mucosa and muscularis mucosae to be evidence low-grade malignant mucinous epithelium in the perito- of low-grade appendiceal mucinous neoplasms (LAMNs; neum (ie, pseudomyxoma peritonei). Opponents of the lat- Figure 10.3E). Adenomas generally lack complex architec- ter argue that although application of this terminology to tural features. They frequently contain flat or undulating such a broad spectrum of lesions, ranging from those that epithelium, but villous projections are present in many are clearly benign to those that behave in a malignant fash- cases. The crypts have straight luminal edges similar to ion, makes classification easier, its clinical utility is limited. those of the non-neoplastic appendix. Some lesions con- In our practice, we use the term “low-grade appendi- tain crypts with a serrated appearance near the lumen. ceal mucinous neoplasm” to describe situations in which Most adenomas display low-grade cytologic atypia there is uncertainty regarding biologic risk, namely those characterized by nuclear hyperchromasia and pseudostrat- cases containing mucin pools (but no neoplastic epithe- ification with rare mitotic figures. High-grade dysplasia is lium) that transgress the muscularis mucosae and extend uncommon, and is characterized by architectural abnor- into the appendiceal wall or onto the serosal surface, but malities, including cribriform and micropapillary growth, are confined to the right lower quadrant. We classify lesions as well as nuclear pleomorphism with loss of cell polarity entirely confined to the mucosa as mucinous adenomas, and and readily identifiable mitotic figures. Appendiceal muci- those with peritoneal tumor deposits as low-grade muci- nous adenomas are cured by appendectomy regardless nous adenocarcinomas. In our experience, the overwhelm- of the degree of dysplasia. However, some authors have ing majority of appendiceal mucinous tumors are amenable proposed that adenoma at the surgical resection margin to this classification scheme, which allows one to clearly warrants concern for recurrent disease or metastasis, and convey information to the clinicians regarding biologic risk. consider such tumors to have uncertain malignant poten- However, as mentioned in the previous section, accurate tial or low risk for recurrence. Submission of the entire classification depends on adequate gross evaluation and appendix is recommended

in cases of mucinous adenoma, sectioning as well as careful histologic examination. in order to exclude the possibility of extra-appendiceal Mucinous neoplasms that transgress the muscularis epithelium and/or mucin. mucosae and show high-grade cytology are classified as high- grade mucinous adenocarcinomas, regardless of growth Low-Grade Appendiceal Mucinous Neoplasm pattern, and should never be considered in the spectrum of LAMN. High-grade lesions infrequently produce the clinical Low-grade appendiceal mucinous neoplasm (LAMN) is a appearance of pseudomyxoma peritonei and are less likely term applied to appendiceal mucinous tumors that show to respond to cytoreductive surgery and chemotherapy. Of features portending increased risk of peritoneal dissemina- note, both low- and high-grade tumors that extend beyond tion. They may appear grossly unremarkable or produce the muscularis mucosae are staged as adenocarcinomas in a cystically dilated appendix filled with tenacious mucin, the seventh edition of the American Joint Committee on resembling a mucinous (cyst)adenoma (Figure 10.4A–C). Cancer (AJCC) Staging Manual. In other words, cases clas- Grossly visible mucin on the serosal surface is an impor- sified as either LAMN or well-d ifferentiated mucinous car- tant finding and, if present, should be submitted entirely cinoma are similarly staged in the TNM system. for histologic evaluation to document the presence, or Mucinous tumors associated with acellular mucin con- absence, of extra-appendiceal neoplastic epithelium. fined to the right lower quadrant have an extremely low Appendices that are encased in mucin, or essentially risk of progression to pseudomyxoma peritonei, whereas replaced by mucinous neoplasia, are highly likely to rep- one-third of patients with any amount of neoplastic epi- resent mucinous adenocarcinomas associated with perito- thelium outside the appendix develop peritoneal dissemi- neal tumor deposits. nation that can result in the death of the patient. For this The histologic features of LAMNs are poorly defined. reason, low-grade appendiceal mucinous neoplasms with Some authors use this terminology to describe lesions lim- periappendiceal mucin should be submitted entirely for ited to the luminal epithelium of the appendix that show histologic evaluation, in order to exclude the presence of mucosal alterations, such as atrophy or fibrosis of the extra-appendiceal neoplastic epithelium. Management of mucosa and muscularis mucosae with hyalinization or cal- patients with extra-appendiceal mucin limited to the peri- cification (Figure 10.4D). This term has also been used to appendiceal region is controversial. They should undergo 10 Neoplasms of the Appendix 2 5 3 (A) (B) (C) (D) (E) FIGURE 10.3 Mucinous cystadenomas cause localized (A) or fusiform (B–C) dilatation of the appendix (C, courtesy of Dr. George Gray). This adenoma has circumferential villous architecture and is surrounded by an intact muscularis mucosae (D). Higher magnification reveals diminished lamina propria and lymphoid tissue overlying the muscularis mucosae (arrow). The submucosa is slightly cellular and fibrotic (E). 2 5 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 10.4 A low-grade appendiceal mucinous neoplasm distends the appendix (arrow), but is not associated with extra- appendiceal mucin (A). Opening the appendix of the same case reveals copious amounts of tenacious mucin (B). Cross sections through another mucinous tumor reveal distention of the appendix by thick mucinous secretions (C). Although one may consider a diagnosis of either mucinous (cyst)adenoma or low-grade mucinous neoplasm based on these features, careful examination of the pericolic fat reveals mucinous nodules in the colonic mesentery (arrow), and additional biopsies from the peritoneum reveal disseminated disease. Thus, this lesion is best classified as a low-grade mucinous adenocarcinoma. Sections through the wall of a low-grade appendiceal neoplasm demonstrate extensive denudation of the surface epithelium with pools of dissecting mucin in the wall. The muscularis propria is largely effaced and replaced by fibrosis with lymphoid aggregates (D). radiographic surveillance to evaluate for development of develop secondary to the spread of low-grade mucinous mucinous ascites, but there are no data to support cyto- tumors of the appendix, although rare cases are associated reductive surgery with intraperitoneal chemotherapy as a with origins from other sites (eg, ovary). Mucin accumu- preventive measure, and right colectomy offers no survival lates in dependent areas, including the greater omentum, benefit over appendectomy alone. under the right hemidiaphragm, behind the liver, and left abdominal gutter and pelvis (Figure 10.6A). Patients are Mucinous Adenocarcinoma and usually older adults, and women are reportedly affected more frequently than men, although this difference may Pseudomyxoma Peritonei reflect a reporting bias in relatively small series of patients Pseudomyxoma peritonei is a descriptive term denoting presenting to gynecologic oncologists with ovarian mucin accumulation in the peritoneal cavity. Most cases involvement. 10 Neoplasms of the Appendix 2 5 5 (A) (B) (C) (D) FIGURE 10.5 This low-grade appendiceal mucinous neoplasm is lined by undulating epithelium that rests on fibrotic connective tissue without an apparent lamina propria or muscularis mucosae. Dissecting mucin is present subjacent to the epithelium (arrow), which shows low-grade cytologic features (A). Another mucinous neoplasm is associated with large mucin pools in the appendiceal wall (B). This same mucinous tumor also displays round mucin pools confined to the appendiceal wall (C). The pools contain strips of low-grade epithelium that warrant a diagnosis of at least low-grade appendiceal mucinous neoplasm, although the AJCC classifies this type of lesion as a carcinoma for staging purposes (D). Peritoneal tumor deposits are morphologically simi- High-grade features also include solid cell clusters that dis- lar to the primary appendiceal tumor in most cases. Low- play nuclear enlargement and hyperchromasia, single cell grade lesions consist of mucin pools containing scant necrosis and mitotic activity (Figure 10.6F). Single infil- strips of mucinous epithelium with mild cytologic atypia trating signet ring cells are believed to be a poor prognos- and a paucity of mitotic activity (Figure 10.6B–C). The tic factor and, if present, should be specifically mentioned. background tissue is abnormal, frequently showing hya- Peritoneal deposits that show high-grade features with a linized bands of collagen and reactive fibrosis around desmoplastic tissue response (Figure 10.6E) and lymph mucin pools. node metastases only rarely represent metastases arising Although distinguishing criteria between low- and high- from low-grade mucinous appendiceal tumors, although grade neoplasms are not well-defined, tumors that show progression from low-grade to high-grade cytologic atypia more abundant epithelium in mucin pools or destructive does occur in patients who develop multiple tumor recur- tissue invasion with infiltrative tumor cells should be classi- rences or those who fail treatment. Indeed, the presence of fied as high-grade mucinous carcinomas (Figure 10.6D–F). high-grade epithelial atypia in peritoneal deposits should 2 5 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 10.6 Low-grade appendiceal mucinous adenocarcinomas spread to the peritoneal cavity in the form of mucinous ascites (ie, pseudomyxoma peritonei). They have a tendency to spare the bowel surfaces early in the course of disease, but commonly affect the omentum, producing confluent mucinous nodules (A). Although the criteria used to distinguish between low- and high-grade neoplasms are not well-defined, low-grade lesions should contain rare strips of epithelium (arrow) in otherwise paucicellular mucin pools (B). Lesional cells are polarized with abundant cytoplasm and small nuclei, without appreciable mitotic activity (C). Tumors that show more abundant epithelium in mucin pools (D) or destructive tissue invasion with infiltrative tumor cells and glands (E) are best classified as mucinous carcinomas. High-grade features include solid cell clusters that display nuclear enlargement and hyperchromasia, single cell necrosis (arrow), and mitotic activity (F). 10 Neoplasms of the Appendix 2 57 lead one to suspect a nonappendiceal origin, such as the Diverticula are frequently encountered in association with small bowel, colon, or pancreas. Alternatively, such lesions mucinous adenomas, potentially reflecting increased intra- may represent peritoneal metastases from nonmucinous luminal pressures resulting from inspissated mucin. When appendiceal carcinomas. they occur in association with mucinous adenomas, divertic- Most patients with peritoneal mucinous adenocar- ula can be confused with the “pushing front” of a low-grade cinoma develop multiple tumor recurrences that require appendiceal neoplasm, particularly if they are colonized by several interventions and lead to extensive adhesions that neoplastic epithelium. Features suggesting a diverticulum, prohibit further abdominal therapy. The natural history of rather than a LAMN, include the presence of non- neoplastic the disease is dictated by tumor grade and pathologic stage. epithelial cells and/or lamina propria in the appendiceal wall Patients with low-grade mucinous adenocarcinoma in the or subserosa, continuity of mural epithelium with that lin- peritoneum have 5- and 10-year survival rates of 75% and ing the lumen, and an intact, normal-appearing muscularis 68%, compared to approximately 55% and 13%, respec- propria. Denuded diverticula may appear as solitary mucin tively, for high-grade carcinomas. Aggressive modern ther- pools associated with lamina propria elements and variable apeutic approaches include a combination of surgery and inflammatory changes. intra-abdominal chemotherapy. Surgical peritonectomy with Appendiceal mucinous adenomas may be associated resection of multiple organs (eg, omentum, spleen, gallblad- with rupture and extrusion of mucin onto the serosal sur- der, some, or all of the affected stomach, colon, and small face, in which case a diagnosis of LAMN may be consid- bowel, uterus, ovaries, and fallopian tubes) is aimed at com- ered. Ruptured adenomas generally show a single focus plete tumor cytoreduction. Chemotherapy is in the form of of transmural extra-appendiceal mucin associated with hyperthermic intra-operative intraperitoneal chemotherapy attenuation of the appendiceal wall, striking inflammation (HIPEC) supplemented by additional cycles of early postop- and organizing granulation tissue at the serosal surface, erative intraperitoneal chemotherapy (EPIC). Five-year sur- and an absence of neoplastic epithelium on the serosa or vival rates of 86% are achievable among low-grade tumors in the appendiceal wall deep to the muscularis mucosae. that are treated with cytoreductive surgery and chemother- Multifocal or extensive transgression of the muscularis apy compared to 50% for high-grade tumors. Complete mucosae by mucin pools, copious amounts of mucin on cytoreduction improves survival compared to cases in which the serosal surface, mucin beyond the right lower quad- gross residual disease is left behind following surgery. rant, and extra-appendiceal neoplastic epithelium should be considered evidence of a LAMN at a minimum, and Ancillary Studies some would classify such a lesion as low-grade mucinous adenocarcinoma. Cases classified as “ruptured” adenomas Appendiceal mucinous neoplasms and pseudomyxoma should be submitted entirely for histologic evaluation and peritonei usually show immunoexpression of cytokeratin designated with a comment implying a low risk of recurrent (CK)20, CDX2, and MUC2, and up to 40% coexpress disease and a need for clinical follow-up as appropriate. CK7. Appendiceal adenomas and mucinous adenocarci- nomas show KRAS mutations in approximately 50% of cases, but do not harbor abnormalities in BRAF, APC, or NONMUCINOUS APPENDICEAL NEOPLASMS DCC, or show microsatellite instability. Carcinomas may also have loss of heterozygosity or chromosome 18q muta- Overview tions affecting DPC4, a tumor suppressor gene. Nonmucinous adenomas of the appendix generally resemble their colonic counterparts in that they display Differential Diagnosis of Appendiceal tubular, villous, or serrated crypt architecture. Most are Mucinous Neoplasms asymptomatic and, thus, reporting bias in the literature is heavily skewed toward appendectomy specimens removed Several lesions that have no biologic risk can cause diag- for symptoms of appendicitis and colectomy specimens nostic confusion with appendiceal mucinous neoplasms. obtained for unrelated reasons. Invasive nonmucinous Some appendices contain mucosal hyperplasia that can be adenocarcinomas of the appendix are not well described, misinterpreted as a neoplasm, particularly when associated but generally arise in association with serrated neoplasms with other findings, such as mucus retention or diverticula. and goblet cell carcinoid tumors. Nonmucinous adenocar- Non-neoplastic hyperplasia contains a mixed population of cinomas involving the proximal appendix often represent mucin-containing columnar epithelial cells without cytologic extension of a colonic tumor into the appendix. atypia or appreciable mitotic activity. Diverticula represent herniations of mucosa through the muscularis propria and Colonic-Type Adenomas of the Appendix are best appreciated at low magnification (Figure 10.7A–D). They may be solitary or multiple and occur in association Colonic-type adenomas of the appendix are uncom- with mucinous adenomas or in non-neoplastic appendices. mon, and lesions involving the proximal appendix likely 2 5 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 10.7 The differential diagnosis of appendiceal mucinous neoplasia includes appendiceal diverticulosis either with, or without, an associated mucinous neoplasm. Diverticula represent herniations of mucosa and submucosa through the muscularis propria. The diagnosis is straightforward when diverticula contain non-neoplastic epithelium (A), but can be challenging when diverticula are colonized by mucinous neoplasia (arrow) that simulates the appearance of a low-grade appendiceal mucinous

neoplasm (B). The presence of a lamina propria investing the epithelium of this diverticulum is a helpful feature indicating that this is a cystadenoma rather than a LAMN (C). Some diverticula rupture and are associated with inflammatory changes, including fibrosis, mucin pools, and lymphoid aggregates (D). Although one may be concerned that such findings reflect a mucinous neoplasm, the luminal epithelium of this case was essentially normal. represent extension of colonic adenomas into the appen- but form polypoid projections that are sharply demarcated diceal orifice. They are also encountered in appendices from adjacent normal-appearing mucosa. They contain removed with colectomy specimens from patients with crowded, tubular crypts lined by dysplastic epithelial cells familial adenomatous polyposis. Colonic-type adenomas with elongated, hyperchromatic nuclei, similar to tubular do not circumferentially involve the appendiceal mucosa, and villous adenomas of the colon (Figure 10.8). 10 Neoplasms of the Appendix 2 5 9 Non-dysplastic serrated polyps of the appendix show strong immunopositivity for MUC6 and asymmetric Ki-67 labeling of crypt epithelial cells, similar to colorec- tal serrated polyps. They also display decreased staining for DNA repair proteins in the superficial epithelium, reflecting its nonproliferative nature, but staining is pre- served at the crypt bases and the lesions are mismatch proficient by polymerase chain reaction (PCR). Mutually exclusive BRAF and KRAS mutations are detected in approximately 50% and 20% of appendiceal sessile ser- rated polyps, respectively, whereas BRAF mutations are far more common and KRAS mutations are less frequent among similar-appearing colonic polyps. Serrated polyps may precede the development of invasive adenocarcinomas. Most of these adenocarcinomas are asymptomatic until they penetrate the visceral perito- FIGURE 10.8 This colonic-type adenoma forms a neum and cause abdominal pain, so they tend to be locally sessile plaque in the appendix of this patient with familial advanced at the time of diagnosis. Adenocarcinomas asso- adenomatous polyposis. The lesion is sharply demarcated ciated with serrated lesions show a destructive growth from the adjacent mucosa and contains crowded, variably pattern with infiltrative glands that may have a tubular dilated crypts lined by low-grade dysplastic epithelium. or serrated appearance. Some tumors contain abundant extracellular mucin, but they are typically more cellular and show high-grade cytologic features and desmopla- sia beyond the spectrum of changes seen in appendiceal Serrated Lesions of the Appendix mucinous neoplasms that cause pseudomyxoma peritonei. Distinction from the latter is important, as nonmucinous Serrated appendiceal neoplasms show a spectrum of his- carcinomas of the appendix pursue an aggressive clinical tologic features paralleling those of serrated colorectal course and are unlikely to respond to cytoreductive sur- polyps. Hyperplastic polyps are sessile nodules that do gery and heated intraperitoneal chemotherapy. not involve the entire luminal circumference. They are reminiscent of “microvesicular” hyperplastic polyps of Goblet Cell Carcinoid Tumor the colorectum and contain a dual population of goblet cells and nongoblet columnar cells with microvesicles of Most goblet cell carcinoid tumors (also known as crypt cell cytoplasmic mucin. Sessile serrated polyps (also termed carcinomas, among other names) are incidentally discov- sessile serrated adenoma) circumferentially involve the ered in patients with symptoms suggesting acute appen- mucosa and contain elongated crypts with a serrated dicitis, and the diagnosis is rarely suspected at the time of appearance (Figure 10.9A). Similar to colonic lesions, surgery. Goblet cell carcinoid tumors are virtually unique those of the appendix contain dilated crypts that display to the vermiform appendix and show features of both lateral branching or budding and mild cytologic atypia neuroendocrine and epithelial differentiation. Unlike clas- (Figure 10.9B). sic carcinoid tumors, goblet cell carcinoid tumors occur Dysplastic serrated polyps of the appendix resemble anywhere in the appendix but are more common in the their colonic counterparts with conventional cytologic midportion, producing an ill-defined mural thickening dysplasia, and are much less common than appendiceal that is difficult to identify at the time of gross examina- hyperplastic polyps and sessile serrated adenomas with- tion (Figure 10.10A). Aggregates of tumor cells resembling out conventional dysplasia. Some contain crypts lined by abortive colonic crypts permeate the deep mucosa. Cells cells with abundant eosinophilic cytoplasm and pencil- infiltrate the appendiceal wall in nests, cords, and clusters late nuclei (Figure 10.9C), resembling traditional serrated arranged circumferentially around the lumen. The tumor adenomas of the left colon. Nuclei have smooth contours cells are usually associated with a densely collagenous and small nucleoli and mitotic figures are infrequent. The stroma (Figure 10.10B). Tumor cells are distended with cytologic dysplasia in appendiceal sessile serrated polyps large cytoplasmic mucin vacuoles that compress the nuclei is often focal, and can display a tubular, villous, or ser- (Figure 10.10C). Mitotic activity is generally inconspicu- rated appearance. Some dysplastic serrated polyps contain ous and Ki-67 immunolabeling indices are low (less than crypts that architecturally resemble those of a sessile ser- 2%). Goblet cell carcinoid tumors show divergent differen- rated polyp, but are lined by dysplastic epithelium, often tiation with patchy immunopositivity for neuroendocrine with high-grade cytologic features (Figure 10.9D). markers and strong, diffuse mucicarmine positivity. 2 6 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 10.9 Serrated appendiceal lesions share morphologic features with their colonic counterparts. Sessile serrated polyps circumferentially surround the appendiceal lumen (A) and contain elongated crypts that show dilatation and branching in the deep mucosa (B). Some serrated polyps with conventional cytologic dysplasia contain serrated crypts lined by cells with enlarged, hyperchromatic nuclei and abundant eosinophilic cytoplasm, similar to those of a (traditional) serrated adenoma (C). They may show high-grade cytologic features with increased mitotic activity and apoptotic debris (D). At least 50% of goblet cell carcinoid tumors contain although mucinous, intestinal, and poorly differentiated areas of adenocarcinoma, which have been called “adeno- areas are also encountered (Figure 10.10D–F). Features carcinoma ex goblet cell carcinoid.” Approximately half suggestive of carcinoma include nuclear atypia, the pres- of these cases cause symptoms related to an abdominal ence of single infiltrating cells, loss of mucin production, mass, which may reflect the enlarged appendix, or metas- and increased Ki-67 immunolabeling. Cases in which gob- tases to other organs, such as the ovary. The carcinoma- let cell carcinoid tumors are identified in initial sections tous components may display signet ring cell morphology, should be submitted entirely to exclude the possibility of 10 Neoplasms of the Appendix 2 61 (A) (B) (C) (D) (E) (F) FIGURE 10.10 This goblet cell carcinoid tumor shows subtle features on gross examination. The muscularis propria is obliterated by a tumor in the mid-appendix that infiltrates periappendiceal fat (A). The tumor consists of nests of goblet cells embedded in densely collagenous stroma that lacks the cellularity seen in typical desmoplasia (B). Goblet cell carcinoid tumors are cytologically bland and contain crescentic nuclei with minimal mitotic activity. This lesion expands the deep mucosa (C). This adenocarcinoma ex goblet cell carcinoid contains poorly formed nests of goblet cells and single infiltrating signet ring cells. Perineural invasion is present (D). Another adenocarcinoma ex goblet cell carcinoid contains nests of goblet cells and sheets of poorly differentiated malignant cells (E). Adenocarcinomas associated with goblet cell carcinoid tumors may also display mucinous differentiation, as in this case, where small, round pools of extracellular mucin contain nests and tubules of malignant cells (F). concomitant carcinoma. Carcinomas derived from goblet metastasize to regional lymph nodes, peritoneum, and ova- cell carcinoid tumors do not show alterations affecting ries. Both the World Health Organization and American β-catenin, KRAS, DPC4, or TP53 typical of malignan- Joint Commission on Cancer classify and stage goblet cell cies of the lower gastrointestinal tract. carcinoid tumors as adenocarcinomas. Management of Most goblet cell carcinoid tumors deeply invade the goblet cell carcinoid tumors is not standardized, although appendiceal wall to penetrate the serosa, and frequently appendectomy alone is unlikely to be adequate therapy 2 6 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide owing to their high metastatic potential. Surgical man- Ca rr NJ, Sobin LH. Neuroendocrine tumors of the appendix. Semin agement generally consists of right colectomy with lymph Diagn Pathol. 2004;21(2):108–119. Hemminki K, Li X. Incidence trends and risk factors of carcinoid node staging. Extra-appendiceal tumor deposits are more tumors: a nationwide epidemiologic study from Sweden. Cancer. likely to contain carcinomatous elements, in which case the 2001;92(8):2204–2210. natural history is dictated by the tumor grade and stage. In’t Hof KH, van der Wal HC, Kazemier G, Lange JF. Carcinoid tumour of the appendix: an analysis of 1,485 consecutive emergency appen- dectomies. J Gastrointest Surg. 2008;12(8):1436–1438. SUMMARY AND CONCLUSIONS Komminoth P, Arnold R, Capella C, et al. Neuroendocrine Neoplasms of the Appendix. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. WHO Classification of Tumours of the Digestive System. Appendiceal tumors are uncommon lesions that show 4th ed. Lyon: International Agency for Research on Cancer; unique clinicopathologic features. Most neuroendocrine 2010:126–128. tumors are small, incidentally discovered tumors of the Matsukuma KE, Montgomery EA. Tubular carcinoids of the appen- dix: the CK7/CK20 immunophenotype can be a diagnostic pitfall. distal appendix that are adequately managed by appen- J Clin Pathol. 2012;65(7):666–668. dectomy alone, whereas larger tumors may require more Murray SE, Lloyd RV, Sippel RS, Chen H, Oltmann SC. Postoperative extensive surgery with lymph node dissection due to surveillance of small appendiceal carcinoid tumors. Am J Surg. increased metastatic potential. These tumors should be 2014;207(3):342–345. Plockinger U, Couvelard A, Falconi M, et al. Consensus guide- assessed for grade and stage using Ki-67 immunohisto- lines for the management of patients with digestive neuroen- chemistry and criteria enumerated in the AJCC Cancer docrine tumours: well-differentiated tumour/carcinoma of the Staging Manual, 7th Edition. Appendiceal mucinous neo- appendix and goblet cell carcinoma. Neuroendocrinology. plasms are also subject to new grading and staging criteria 2008;87(1):20–30. Williams GT. Endocrine tumours of the gastrointestinal tract-selected aimed at predicting biologic behavior. Tumors confined topics. Histopathology. 2007;50(1):30–41. to the mucosa are classified as adenomas and cured by appendectomy alone, whereas those that extend into the appendiceal wall or periappendiceal soft tissue may spread Mucinous Tumors to the peritoneal cavity and, thus, should be classified as Bradley RF, Stewart JH 4th, Russell GB, Levine EA, Geisinger KR. LAMNs or mucinous adenocarcinoma in order to imply Pseudomyxoma peritonei of appendiceal origin: a clinicopathologic biologic risk. Mucinous tumor deposits in the peritoneum analysis of 101 patients uniformly treated at a single institution, (ie, pseudomyxoma peritonei) may respond to cytoreduc- with literature review. Am J Surg Pathol. 2006;30(5):551–559. tive surgery in combination with intraperitoneal chemo- Chua TC, Al-Zahrani A, Saxena A, et al. Secondary cytoreduction and perioperative intraperitoneal chemotherapy after initial debulk- therapy, particularly if they contain low-grade neoplastic ing of pseudomyxoma peritonei: a study of timing and the impact epithelium. Most nonmucinous carcinomas of the appen- of malignant dedifferentiation. J Am Coll Surg. 2010;211(4): dix are derived from serrated appendiceal neoplasms and 526–535. goblet cell carcinoid tumors. They share some morphologic Davison JM, Hartman DA, Singhi AD, et al. Loss of SMAD4 protein expression is associated with high tumor grade and poor progno- and molecular features with their colonic counterparts, sis in disseminated appendiceal mucinous neoplasms. Am J Surg including frequent BRAF or KRAS mutations. Goblet cell Pathol. 2014;38(5):583–592. carcinoid tumors frequently behave in a more aggressive Elias D, Gilly F, Quenet F, et al. Pseudomyxoma peritonei: a French fashion than classic neuroendocrine tumors and may give multicentric study of 301 patients treated with cytoreductive surgery and intraperitoneal chemotherapy. Eur J Surg Oncol. rise to high-grade carcinomas that behave in a stage depen- 2010;36(5):456–462. dent fashion. For this reason, they are classified as adeno- Fish R, Selvasekar C, Crichton P, et al. Risk-reducing laparoscopic cyto- carcinomas, rather than neuroendocrine tumors. reductive surgery and hyperthermic intraperitoneal chemotherapy for low-grade appendiceal mucinous neoplasm: early outcomes and technique. Surg Endosc. 2014;28(1):341–345. Foster JM, Gupta PK, Carreau JH, et al. Right hemicolectomy is SELECTED REFERENCES not routinely indicated in pseudomyxoma peritonei. Am Surg. 2012;78(2):171–177. General Gonzalez-Moreno S, Sugarbaker PH. Right hemicolectomy does not confer a survival advantage in patients with mucinous carcinoma Appendix. In: Edge, S, Byrd, D, Compton, CC, Fritz, A, Green, F, of the appendix and peritoneal seeding. Br J Surg. 2004;91(3): Trotti, A, eds. AJCC Cancer Staging Manual. 7th ed. New York: 304–311. Springer; 2009:133–141. Jarvinen P, Jarvinen HJ, Lepisto A. Survival of patients with pseu- Carr NJ, Sobin LH. Tumors of the appendix. In: Bosman FT, Carneir F, domyxoma peritonei treated by serial debulking. Col Dis. Hruban RH, Theise ND, eds. WHO Classification

of Tumours 2010;12(9):868–872. of the Digestive System. 4th ed. Lyon: International Agency for Kabbani W, Houlihan PS, Luthra R, et al. Mucinous and nonmu- Research on Cancer (IARC); 2010:122–125. cinous appendiceal adenocarcinomas: different clinicopatho- logical features but similar genetic alterations. Mod Pathol. Neuroendocrine Tumors 2002;15(6):599–605. Lamps LW, Gray GF Jr, Dilday BR, Washington MK. The coexistence B urke AP, Sobin LH, Federspiel BH, Shekitka KM. Appendiceal carci- of low-grade mucinous neoplasms of the appendix and appendiceal noids: correlation of histology and immunohistochemistry. Mod diverticula: a possible role in the pathogenesis of pseudomyxoma Pathol. 1989;2(6):630–637. peritonei. Mod Pathol. 2000;13(5):495–501. 10 Neoplasms of the Appendix 2 6 3 Miner TJ, Shia J, Jaques DP, et al. Long-term survival following treat- Carr NJ, McCarthy WF, Sobin LH. Epithelial noncarcinoid tumors ment of pseudomyxoma peritonei: an analysis of surgical therapy. and tumor-like lesions of the appendix. A clinicopathologic study Ann Surg. 2005;241(2):300–308. of 184 patients with a multivariate analysis of prognostic factors. Misdraji J, Burgart LJ, Lauwers GY. Defective mismatch repair in the Cancer. 1995;75(3):757–768. pathogenesis of low-grade appendiceal mucinous neoplasms and Cortina R, McCormick J, Kolm P, Perry RR. Management and prog- adenocarcinomas. Mod Pathol. 2004;17(12):1447–1454. nosis of adenocarcinoma of the appendix. Dis Colon Rectum. Misdraji J, Yantiss RK, Graeme-Cook FM, et al. Appendiceal mucinous 1995;38(8):848–852. neoplasms: a clinicopathologic analysis of 107 cases. Am J Surg Gui X, Qin L, Gao ZH, Falck V, Harpaz N. Goblet cell carcinoids at Pathol. 2003;27(8):1089–1103. extraappendiceal locations of gastrointestinal tract: an underrec- Pai RK, Beck AH, Norton JA, Longacre TA. Appendiceal mucinous ognized diagnostic pitfall. J Surg Onc. 2011;103(8):790–795. neoplasms: clinicopathologic study of 116 cases with analysis Holt N, Gronbaek H. Goblet cell carcinoids of the appendix. Scientific of factors predicting recurrence. Am J Surg Pathol. 2009;33(10): World Journal. 2013;2013:543696. 1425–1439. Maru D, Wu TT, Canada A, et al. Loss of chromosome 18q and DPC4 Ronnett BM, Yan H, Kurman RJ, et al. Patients with pseudomyxoma peri- (Smad4) mutations in appendiceal adenocarcinomas. Oncogene. tonei associated with disseminated peritoneal adenomucinosis have a 2004;23(3):859–864. signifi cantly more favorable prognosis than patients with peritoneal Moran B, Baratti D, Yan TD, Kusamura S, Deraco M. Consensus state- mucinous carcinomatosis. Cancer. 2001;92:85–91. ment on the loco-regional treatment of appendiceal mucinous neo- Ronnett BM, Zahn CM, Kurman RJ, et al. Disseminated peritoneal adeno- plasms with peritoneal dissemination (pseudomyxoma peritonei). mucinosis and peritoneal mucinous carcinomatosis. A clinicopatho- J Surg Onc. 2008;98(4):277–282. logic analysis of 109 cases with emphasis on distinguishing pathologic Pai RK, Hartman DJ, Gonzalo DH, et al. Serrated lesions of the appen- features, site of origin, prognosis, and relationship to “pseudomyx- dix frequently harbor KRAS mutations and not BRAF mutations oma peritonei.” Am J Surg Pathol. 1995;19(12):1390–1408. indicating a distinctly different serrated neoplastic pathway in the Schomas DA, Miller RC, Donohue JH, et al. Intraperitoneal treatment appendix. Hum Pathol. 2014;45(2):227–235. for peritoneal mucinous carcinomatosis of appendiceal origin after Palanivelu C, Rangarajan M, Annapoorni S, et al. Laparoscopic right operative management: long-term follow-up of the Mayo Clinic hemicolectomy for goblet-cell carcinoid of the appendix: report of experience. Ann Surg. 2009;249(4):588–595. a rare case and literature survey. J Laparoendosc Adv Surg Tech A. Smeenk RM, van Velthuysen ML, Verwaal VJ, Zoetmulder FA. 2008;18(3):417–421. Appendiceal neoplasms and pseudomyxoma peritonei: a popula- Renshaw AA, Kish R, Gould EW. Sessile serrated adenoma is associated tion based study. Eur J Surg Oncol. 2008;34(2):196–201. with acute appendicitis in patients 30 years or older. Am J Clin Smeenk RM, Verwaal VJ, Antonini N, Zoetmulder FA. Survival analy- Pathol. 2006;126(6):875–877. sis of pseudomyxoma peritonei patients treated by cytoreductive Rubio CA. Serrated adenomas of the appendix. J Clin Pathol. surgery and hyperthermic intraperitoneal chemotherapy. Ann 2004;57(9):946–949. Surg. 2007;245(1):104–109. Stancu M, Wu TT, Wallace C, et al. Genetic alterations in goblet cell Yantiss RK, Shia J, Klimstra DS, et al. Prognostic significance of local- carcinoids of the vermiform appendix and comparison with gastro- ized extra-appendiceal mucin deposition in appendiceal mucinous intestinal carcinoid tumors. Mod Pathol. 2003;16(12):1189–1198. neoplasms. Am J Surg Pathol. 2009;33:248–255. Sugarbaker PH, Chang D. Results of treatment of 385 patients with Youssef H, Newman C, Chandrakumaran K, et al. Operative findings, peritoneal surface spread of appendiceal malignancy. Ann Surg early complications, and long-term survival in 456 patients with Onc. 1999;6(8):727–731. pseudomyxoma peritonei syndrome of appendiceal origin. Dis Col Sugarbaker PH, Jablonski KA. Prognostic features of 51 colorectal and Rect. 2011;54(3):293–299. 130 appendiceal cancer patients with peritoneal carcinomatosis Zauber P, Berman E, Marotta S, Sabbath-Solitare M, Bishop T. treated by cytoreductive surgery and intraperitoneal chemother- Ki-ras gene mutations are invariably present in low-grade muci- apy. Ann Surg. 1995;221(2):124–132. nous tumors of the vermiform appendix. Scand J Gastroenterol. Tang LH, Shia J, Soslow RA, et al. Pathologic classification and clini- 2011;46(7–8):869–874. cal behavior of the spectrum of goblet cell carcinoid tumors of the appendix. Am J Surg Pathol. 2008;32(10):1429–1443. Nonmucinous Tumors van Eeden S, Offerhaus GJ, Hart AA, et al. Goblet cell carcinoid of the appendix: a specific type of carcinoma. Histopathology. Alsaad KO, Serra S, Schmitt A, Perren A, Chetty R. Cytokeratins 7 and 2007;51(6):763–773. 20 immunoexpression profile in goblet cell and classical carcinoids Yajima N, Wada R, Yamagishi S, et al. Immunohistochemical expres- of appendix. Endocr Pathol. 2007;18(1):16–22. sions of cytokeratins, mucin core proteins, p53, and neuroendo- Bellizzi AM, Rock J, Marsh WL, Frankel WL. Serrated lesions of the crine cell markers in epithelial neoplasm of appendix. Hum Pathol. appendix: a morphologic and immunohistochemical appraisal. Am 2005;36(11):1217–1225. J Clin Pathol. 2010;133(4):623–632. Yantiss RK, Panczykowski A, Misdraji J, et al. A comprehensive study Burke AP, Sobin LH, Federspiel BH, Shekitka KM, Helwig EB. Goblet of nondysplastic and dysplastic serrated polyps of the vermiform cell carcinoids and related tumors of the vermiform appendix. Am appendix. Am J Surg Pathol. 2007;31(11):1742–1753. J Clin Pathol. 1990;94(1):27–35. Younes M, Katikaneni PR, Lechago J. Association between mucosal Butler JA, Houshiar A, Lin F, Wilson SE. Goblet cell carcinoid of the hyperplasia of the appendix and adenocarcinoma of the colon. appendix. Am J Surg. 1994;168(6):685–687. Histopathology. 1995;26(1):33–37. 11 Neoplasms of the Colon BENJAMIN J. SWANSON, SCOT T R . OW ENS, AND W ENDY L . FR ANKEL INTRODUCTION have some degree of overlapping morphologic findings, and are discussed here because they may occasionally Adenocarcinoma is the most common malignancy arising enter into the differential diagnosis with adenomas and in the colorectum, and colorectal adenocarcinoma (CRC) CRC. is the third most common malignancy overall in the United Patients with mucosal prolapse are often symptom- States. It is estimated that approximately 133,000 people atic, with melena and rectal bleeding among the most will be diagnosed with CRC in 2015, and about 50,000 common complaints. These lesions may present as pol- people will die of the disease. Furthermore, 1.4 million yps, ulcers, or masses, or a combination of those lesions. new cases of CRC were diagnosed worldwide in 2012, Histologic features include perpendicular fibromus- with approximately 694,000 deaths. Although a wide cular hyperplasia and splaying and disorganization of variety of other benign and malignant tumors are seen in the muscularis mucosae/lamina propria interface; pro- the colon, they are much less common; often these lesions liferating, dilated, and thrombosed blood vessels in the present as polyps that are found on screening colonoscopy. lamina propria; mucosal erosion and ulceration with an Similar to the small bowel, there are four major cat- overlying acute inflammatory exudate with abundant egories of primary colorectal malignancies: adenocar- fibrin; reactive epithelial changes such as crypt hyper- cinoma, neuroendocrine tumors (NETs), mesenchymal plasia and serration; and diamond or triangular-shaped tumors, and lymphomas (Table 11.1). The World Health crypts (Figure 11.1A–C). Organization (WHO) 2010 classification for colorectal Diverticular polyps (also known as polypoid prolapsing tumors is shown in Table 11.2. mucosal folds) are similar lesions that develop in associa- tion with diverticular disease. Early in their development, these polyps may contain congested blood vessels, hem- INFLAMMATORY AND orrhage and hemosiderin deposition (Figure 11.2A–C), NON-NEOPLASTIC LESIONS which may impart a red–brown discoloration macro- scopically. Older polyps show histologic findings similar Inflammatory lesions in the colon often present as polyps, to other mucosal prolapse polyps, including fibromuscular which are then endoscopically biopsied or removed. Many hyperplasia and reactive epithelial changes. colonic inflammatory lesions are considered to be within Inflammatory cap polyposis is also considered within the spectrum of mucosal prolapse and related conditions, the spectrum of prolapse polyps. They are usually found in including solitary rectal ulcer syndrome, diverticular pol- the rectosigmoid colon, but occasionally involve the entire yps, colitis cystica profunda, inflammatory cap polyps, colon. Inflammatory cap polyposis appears in two clini- and inflammatory cloacogenic polyps. All are believed to cal settings: patients (usually women) with prolapse-type be caused by traction and twisting of the mucosa from symptoms who have solitary or a few lesions confined to peristalsis-induced trauma, with torsion of blood vessels the rectum, and patients with more extensive disease who and ischemic damage to the mucosa. The polyps included do not have a history of constipation. The most striking in the spectrum of mucosal prolapse-associated lesions all feature is a “cap” of fibrinopurulent exudate (Figure 11.3) 2 6 5 2 6 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 11.1 Clinicopathologic Characteristics of Primary Colorectal Malignancies Neuroendocrine Tumor/ Malignant Mesenchymal Adenocarcinoma Carcinoma B Cell Lymphoma Tumor Relative Incidence (%) 90%–95% 0.4% 0.2%–0.4% Extremely rare Age (median, years) 72 Colon 66 50–70 Variable Rectum 56 Gender (male:female) Colon 1.2:1 Rectum 1.5:1 Colon 0.66:1 2:1 Variable Rectum 1.02:1 Most common site(s) Sigmoid, rectum Rectum, cecum, sigmoid Cecum Variable Source: Data adapted from WHO Classification of Tumours of the Digestive System (2010). TABLE 11.2 WHO 2010 Classification of Tumors of the Colon overlying an eroded or ulcerated surface, often with prom- inent granulation tissue. Crypt hyperplasia and fibromus- Epithelial Tumors Hamartomas cular change of the lamina propria are variably present. Juvenile polyp Colitis cystica profunda most commonly occurs as a Peutz–Jeghers polyp solitary lesion in the rectum, but can be found anywhere Cowden-associated polyp (PTEN hamartomatous syndrome) in the colon, often in association with inflammatory bowel Premalignant Lesions Adenoma disease, ostomy sites, or other conditions that feature muco- Tubular sal ulceration and repair. These lesions often regress after Villous the underlying mechanism of traumatic injury is removed. Tubulovillous Histologically, colitis cystica profunda features misplaced, Dysplasia (intraepithelial neoplasia) Low grade cystically dilated, mucin-filled crypts lined by intestinal High grade epithelium, which are present within the submucosa and Serrated Lesions even the muscularis propria in rare cases (Figure 11.4A–B). Hyperplastic polyp The misplaced crypts are lobular in arrangement and are Sessile serrated adenoma/polyp Traditional serrated adenoma surrounded by lamina propria; there are often associated Carcinoma hemosiderin-laden macrophages as well. The epithelium in Adenocarcinoma the misplaced crypts is not neoplastic. The main differen- Cribriform comedo-type adenocarcinoma tial diagnosis is colorectal adenocarcinoma, both clinically Medullary carcinoma Micropapillary carcinoma and histologically. Invasive adenocarcinoma, however, fea- Mucinous adenocarcinoma tures neoplastic epithelium (with much more significant Serrated adenocarcinoma atypia than the surface epithelium), irregular or angulated Signet ring cell carcinoma glands, and associated desmoplastic stroma. The glands in Adenosquamous carcinoma Spindle cell carcinoma colitis cystica profunda, in contrast, are not angulated, and Squamous cell carcinoma are surrounded by lamina propria; desmoplasia is absent. Undifferentiated carcinoma If there is any cytologic atypia in the context of colitis cys- Neuroendocrine Neoplasms tica profunda, it should be similar in degree to that seen in Neuroendocrine tumor (NET) NET G1 (carcinoid) the overlying surface epithelium. NET G2 Inflammatory cloacogenic polyps are prolapse-type Neuroendocrine carcinoma (NEC) polyps that typically present as a single polypoid mass in Large cell NEC the anterior wall of the anorectum. Histologically, due to Small cell NEC Mixed adenoneuroendocrine carcinoma (MANEC) the anatomic location, these lesions may have colorectal EC cell, serotonin-producing NET columnar mucosa in addition to anal stratified squamous L cell, glucagon-like peptide-producing, and PP/PYY-producing epithelium, or a combination of both. The most striking NET feature of this polyp is the villiform morphology of the Mesenchymal Tumors Leiomyoma surface epithelium (Figure 11.5). Similar to other prolapse- Lipoma type polyps, there may be stromal hyalinization, which is Angiosarcoma most prominent at the base of the polyp, as well as elastin Gastrointestinal stromal tumor deposition, which is unique to this type of polyp. Due to Kaposi sarcoma Leiomyosarcoma the villous growth pattern, these may mimic a villous ade- Lymphomas noma at low power. However, inflammatory cloacogenic Secondary Tumors polyps lack cytologic dysplasia. Inflammatory myoglandular polyps

are rare, usually Source: Data adapted from WHO Classification of Tumours of the Digestive System, 4th Edition, 2010. solitary, and primarily found in the distal colon. Many 11 Neoplasms of the Colon 2 67 (A) (B) (C) FIGURE 11.1 A low-power view of a prolapse polyp shows reactive epithelial changes such as crypt hyperplasia and serration (A). Disorganization of the muscularis mucosae/lamina propria interface and perpendicular fibromuscular hyperplasia are common (B), as are proliferating, dilated, and sometimes thrombosed blood vessels in the lamina propria and mucosal erosion and ulceration. Diamond- or triangular-shaped crypts are often seen at the base of prolapse polyps (C). consider these to be within the spectrum of colonic mucosal surface epithelium is often eroded or ulcerated, with an prolapse polyps because of the significant histologic over- associated fibrinopurulent exudate and reactive epithelial lap. However, inflammatory myoglandular polyps often changes (Figure 11.6). Glands are typically hyperplastic lack the well-formed granulation tissue cap seen in inflam- and/or cystically dilated, and there is often hemosiderin matory cap polyps, the villous architecture of inflamma- deposition in the lamina propria. An older age at presenta- tory cloacogenic polyps, and the misplaced epithelium of tion favors inflammatory myoglandular polyp over Peutz– colitis cystica profunda; in addition, they are not neces- Jeghers polyp, and Peutz–Jeghers polyps typically do not sarily associated with diverticular disease or inflammatory have surface erosions, ulcerations, or marked inflamma- bowel disease. For these reasons, other authorities believe tory epithelial changes. these polyps could represent hamartomas. Myoglandular Although all of the above-mentioned lesions within the polyps have a radially arranged network of muscularis spectrum of mucosal prolapse are reactive, it can be chal- mucosae that may closely resemble Peutz–Jeghers-type lenging to distinguish them from other types of neoplastic polyps; however, in contrast to Peutz–Jeghers polyps, the polyps and even invasive adenocarcinoma. This may be a 2 6 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 11.2 Diverticular polyps occur near the orifices of diverticula, and often have a red–brown macroscopic appearance due to hemorrhage and hemosiderin deposition (A). This low-power view shows dilated and congested blood vessels in the submucosa; the diverticular orifice is to the left (B, arrow). In early diverticular polyps, prominent mucosal hemorrhage and hemosiderin deposition are present (C). particular problem during intraoperative frozen section evaluation, when the splaying and fibromuscular hyperpla- sia with entrapped glands mimic invasive adenocarcinoma. Inflammatory polyps (or pseudopolyps) are another common inflammatory lesion of the colon. These polyps occur as a result of regeneration and repair secondary to mucosal injury, and are present in numerous contexts, including inflammatory bowel disease, ischemic colitis, and infectious colitis. The histologic features of these pol- yps are related to the regenerative biology of the mucosa, and include variable amounts of surface ulceration, granu- lation tissue, crypt distortion, and neutrophilic inflamma- tion that may extend into the crypts (Figure 11.7). The epithelium may exhibit marked atypia due to regenerative and reactive changes, which can be concerning for dyspla- FIGURE 11.3 Inflammatory cap polyps feature a sia. Maturation at the mucosal surface, as well as the pres- prominent “cap” of fibrinopurulent exudate overlying an ence of surrounding inflammation and ulceration, helps ulcerated surface, with marked capillary proliferation. distinguish reactive changes from true dysplasia. 11 Neoplasms of the Colon 2 6 9 (A) (B) FIGURE 11.4 Colitis cystica profunda consists of misplaced mucosal elements, including markedly dilated crypts filled with mucin (A). The misplaced epithelium has a lobular configuration, and is surrounded by lamina propria (B). The misplaced epithelium is not dysplastic, and resembles the overlying surface epithelium, helping to distinguish it from invasive adenocarcinoma. HAMARTOMATOUS POLYPS Juvenile polyps (Figure 11.8A–C) may occur spo- radically or as part of juvenile polyposis syndrome (JPS). Several types of hamartomatous polyps are seen in the Sporadic juvenile polyps are the most common colorectal gastrointestinal tract; these are discussed in detail in polyp in children 10 years of age and younger. Patients Chapter 6. Features pertinent to the colon are briefly men- with JPS have mutations or deletions in the SMAD4/ tioned here. DPC4 or BMPR1A genes. JPS patients have a 68% risk FIGURE 11.5 The most striking feature of inflammatory cloacogenic polyps, which are included in the spectrum FIGURE 11.6 Myoglandular polyps contain well-developed of prolapse polyps, is the villiform morphology of the smooth muscle surrounding lobules of benign epithelium, surface epithelium. Similar to other prolapse polyps, there which may resemble Peutz–Jeghers-type polyps. The may be stromal hyalinization, perpendicular fibromuscular surface epithelium is often eroded or ulcerated, with an hyperplasia, and proliferation of capillaries in the lamina associated fibrinopurulent exudate and reactive epithelial propria. Photograph, courtesy of Dr. Rhonda Yantiss. changes. 270 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Peutz–Jeghers polyps (Figure 11.9) typically occur in patients with Peutz–Jeghers syndrome (PJS), an autosomal dominant disorder caused by mutation to the gene STK11; they rarely occur sporadically. PJS polyps can be found in the stomach, small intestine, and large intestine, and may give rise to dysplasia and carcinoma. Care must be taken not to misdiagnose herniated/misplaced epithelium as invasive adenocarcinoma, particularly if the displaced epithelium is dysplastic. PTEN hamartoma syndrome is an autosomal domi- nant syndrome that encompasses several syndromes that were previously thought to be distinct, including Cowden syndrome, Bannayan–Ruvalcaba–Riley syndrome, and Lhermitte–Duclos disease, among others. All of these disorders have mutations in the tumor suppressor PTEN (phosphatase and tensin homolog) on chromosome 10. FIGURE 11.7 Inflammatory polyps (also known as These patients have hamartomatous gastrointestinal tract inflammatory pseudopolyps) feature inflamed polypoid polyps as well as a variety of extra-intestinal manifesta- colonic mucosa with surface ulceration, prominent tions, and an increased risk of malignancy at various sites granulation tissue, and crypt distortion. including breast, thyroid, and the colon. Patients with this syndrome may also have hyperplastic, inflammatory, ade- nomatous, and ganglioneuromatous polyps in the colon. The hamartomatous polyps in PTEN hamartoma syn- drome are not believed to have neoplastic potential. of developing colorectal cancer by 60 years of age, with a Cronkhite–Canada syndrome is an idiopathic gastro- mean age at diagnosis of 35 years; there is also increased enterocolopathy affecting older adults (see also Chapter 8). risk of small intestinal, gastric, and possibly pancreatic Patients with this disease have diffuse polyposis of the cancer. Syndromic juvenile polyps may also give rise to gastrointestinal tract that spares the esophagus, as well as foci of dysplasia and carcinoma, but these findings are extraintestinal manifestations. The natural history of the very uncommon in sporadic juvenile polyps, and, if identi- disease is variable, but mortality is high. Histologically, fied, should prompt consideration of JPS. the polyps in Cronkhite–Canada syndrome resemble (A) (B) (C) FIGURE 11.8 Juvenile polyps are composed of markedly dilated glands containing inspissated mucin and inflammatory cells, with associated stromal expansion by edema and inflammation (A). This juvenile polyp from a JPS patient shows extensive low-grade dysplasia (B–C). Note that the presence of cystically dilated glands is still detectable. 11 Neoplasms of the Colon 271 criterion of a minimum amount of 25% villous architec- ture for classification as a tubulovillous adenoma is also arbitrary. Due to poor reproducibility and lack of strict definitions, some have questioned the clinical utility of reporting the presence or absence of a villous component, but the supposed significance of a villous component is firmly embedded in the clinical literature. Conventional adenomas of the colon, by defini- tion, contain at least low-grade dysplastic epithelium. Histologically, low-grade dysplasia features pseudostrati- fied, hyperchromatic, pencillate nuclei (Figure 11.10A–G). Prominent large nucleoli are not typically present, and nuclear polarity is maintained with the nuclei predomi- nately restricted to the basal portion of the cells. Mitotic figures and apoptotic bodies are frequently seen. The glands do not form confluent sheets, cribriforming archi- FIGURE 11.9 Peutz–Jeghers polyps have a distinct tecture, or complex budding. The dysplastic changes typi- arborizing architecture featuring lobules of benign cally involve the surface epithelium. colonic epithelium surrounded by prominent bundles of Metaplastic changes can also be found in conven- smooth muscle. The epithelium resembles normal colonic tional adenomas (Figure 11.10D–G). Squamoid metapla- epithelium, with abundant goblet cells (inset). sia is very common, affecting 0.44% of colon adenomas. Histologically, the metaplastic component is composed of monotonous nests of cells that do not have significant nuclear pleomorphism, mitoses, or necrosis. These nests of other hamartomatous polyps, but Cronkhite–Canada can cells are often found at the base of the adenoma, and rarely usually be distinguished based on the clinical presentation have stroma surrounding them that mimics desmoplasia. and the fact that the intervening mucosa shows similar The epithelial cells stain for squamous markers, and are changes to the hamartomatous polyps. sometimes immunoreactive for neuroendocrine markers. For these reasons, it was recently proposed that squamous metaplasia is related or equivalent to an adenoma with a ADENOMATOUS AND SERRATED POLYPS focus of microscopic carcinoid tumor. Salaria et al. dem- onstrated that both the adenomatous component as well Conventional (Intestinal-Type) Adenomas as the microcarcinoid component are positive for nuclear Conventional (intestinal-type) adenomas of the colorec- β-catenin, suggesting that the two components were derived tum are extremely common, as illustrated by the fact that from a common progenitor via the adenomatous polyposis up to 16% of patients in the sixth decade of life have at coli (APC)/Wnt signaling pathway. It remains controversial least one adenoma identified on screening colonoscopy. whether this represents true metaplasia or an alternative The incidence of conventional adenomas increases with differentiation of a stem cell. The main differential diagno- age as well. These adenomas can be classified based on the sis for this entity is an invasive, poorly differentiated carci- degree of villous architecture as well as the degree of dys- noma arising from an adenoma. Close examination of the plasia. Adenomas with less than 25% villous architecture low-grade cytologic features of the squamous metaplasia, are considered tubular adenomas; adenomas with greater as well as the lobular architecture, helps distinguish this than 75% villous architecture are considered villous ade- entity from invasive carcinoma. nomas; and adenomas with 25% to 75% villous formation The second most common type of metaplasia found in are classified as tubulovillous adenomas. Despite these conventional adenomas, (reportedly 0.20%) is Paneth cell definitions, there is high interobserver variability in the metaplasia. This metaplasia is usually not diagnostically quantification of the villous component in adenomatous challenging and should not suggest a diagnosis of inflam- polyps, even among pathologists with interest/experience matory bowel disease (IBD) even if seen in the left colon or in gastrointestinal pathology. Furthermore, the definition rectum. The Paneth cells contain apically located eosino- of “villous” is not particularly well established either. Some philic granules and are similar to those seen normally in authorities have defined a villus, in this context, as a villi- the small bowel and right colon. Other rare metaplasias form structure that is two times higher than the surround- arising in conventional adenomas include clear cell, osse- ing colonic mucosa. However, this definition is arbitrary ous, gastric, and melanocytic metaplasia. and not supported by studies that evaluate the prognostic High-grade dysplasia is characterized by high-grade significance of this morphologic definition. Likewise, the nuclear features and/or high-grade architectural features. 272 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 11.10 (continued) 11 Neoplasms of the Colon 273 (F) (G) FIGURE 11.10 Low-grade adenomatous epithelium in colon polyps is typically composed of cigar-shaped, pseudostratified hyperchromatic nuclei that reach the epithelial surface (A–B). Goblet cells are often decreased. Adenomas with villous architecture have long villi covered by dysplastic epithelium (C). Squamous metaplasia is common in tubular adenomas, which some classify as foci of microcarcinoid based on the immunophenotype (D [arrow], E). Paneth cell metaplasia is common in tubular adenomas (F, arrow). Clear cell change can also be seen in tubular adenomas; this has no clinical significance (G; courtesy Dr. Shawn Kinsey). The nuclei show pleomorphism as well as an increased size of the polyp is more important than the other less nuclear:cytoplasmic ratio (Figure 11.11A–C). The nucleoli reproducible features of an advanced adenoma, and thus may be large with interspersed areas of clear chromatin. they prefer not to mention villous architecture or high- Nuclear polarity is typically lost, and bizarre mitotic fig- grade dysplasia. This opinion, together with concern ures may be seen. Architectural findings include cribri- about overly aggressive therapy for lesions that do not formed glands, confluent sheets of dysplastic

cells, and have metastatic potential, has led some pathologists to complex budding. Most pathologists consider carcinoma refrain from using any other morphologic descriptors for in situ to be within the spectrum of high-grade dysplasia; conventional adenomas (villous, tubular, or high-grade furthermore, most discourage the use of the term carci- dysplasia). Other pathologists continue to use these noma in situ because it is not believed to have the potential descriptors at the request of their colleagues in gastroen- to metastasize, and may lead to confusion and possible terology and surgery. overtreatment. The concept of “advanced” adenomas has arisen Treatment and Surveillance Guidelines in the clinical literature to describe those adenomas that require closer follow-up than typical conventional The current recommendation for screening is to begin at intestinal-type adenomas. Advanced adenomas are char- age 50 for average-risk individuals, with complete removal acterized by at least one of the following features: high- of any adenomas. The endoscopic follow-up for adenoma- grade dysplasia, a villous component, or size greater tous polyps depends on the number, size, and histologic than 1.0 cm. Similar to the debate regarding the repro- characteristics of the polyp. A patient with a small (< 1 ducibility and definitional criteria of villous components cm) tubular adenoma usually has a follow-up colonoscopy in adenomatous polyps, there are similar challenges to in 5 to 10 years, as the risk of neoplasia developing in reporting high-grade dysplasia in adenomatous polyps. a patient with a small tubular adenoma in the Veterans There is poor interobserver agreement, even among Affairs (VA) cooperative study was less than 1% after 5 pathologists with interest/experience in gastrointesti- years. If a patient has greater than three tubular adeno- nal pathology, regarding the designation of high-grade mas, the surveillance interval may be decreased to 3 years; dysplasia, particularly in small (< 1 cm) adenomatous in addition, the typical endoscopic follow-up interval for polyps. In addition, some pathologists believe that the an advanced adenoma is 3 years (rather than 5–10 years). 274 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 11.11 High-grade dysplasia typically shows both architectural complexity (such as cribriform gland architecture, A–B) and high grade cytologic changes including prominent nucleoli and loss of nuclear polarity (C). The risk of developing neoplasia in a polyp of any size the distinction from dysplasia. Close examination of the with high-grade dysplasia, according to the VA cohort adjacent mucosa may also help to identify the epithelial study, is 11% after 5 years. changes as reactive, particularly if they are adjacent to an ulcer or an area of ischemia (Figure 11.12A–B). Finally, Diagnostic Challenges true dysplasia is usually very well demarcated from the surrounding mucosa, whereas reactive changes tend to be Florid reactive epithelial changes can mimic conventional less sharply delineated. adenomas, particularly when the reactive epithelium fea- The distinction between sporadic adenomatous tures nuclear hyperchromasia and stratification, increased polyps and IBD-related polypoid or adenoma-like dys- or atypical mitotic figures, and mucin depletion. However, plasia (also known as dysplasia-associated lesion or reactive/reparative epithelium typically shows maturation mass [DALM]) can be extremely challenging. Patients toward the luminal surface, as opposed to conventional with both ulcerative colitis and Crohn’s disease are at adenomas, which show nuclear hyperchromasia, pseu- increased risk for both dysplasia and eventual progres- dostratification, and mitoses that extend to the mucosal sion to carcinoma. Dysplasia in IBD can present as a surface. When the reactive epithelium is associated with flat, elevated but indistinct lesion, or a discrete polyp- an erosion or ulcer bed, the identification of granula- oid lesion, and can contain either low- or high-grade tion tissue and abundant acute inflammation helps in dysplasia. 11 Neoplasms of the Colon 275 (A) (B) FIGURE 11.12 Reactive/regenerative epithelium near an ulcer (A, arrow; and B) shows nuclear hyperchromasia and stratification that mimics dysplasia. Note the gradual, indistinct transition to clearly benign crypts more distant to the ulcer (A). Histologically, it has been reported in the literature dysplasia arising from the background of IBD. The most that polypoid dysplasia in IBD may contain more inflam- useful histologic finding in these cases often results from mation and chronic mucosal injury than that seen in spo- endoscopic sampling of the mucosa adjacent to the pol- radic adenomas (Figure 11.13A–B). Sporadic adenomas ypoid lesion. In IBD, the mucosa adjacent to the polyp- also tend to have adenomatous glands that populate the oid dysplasia tends to show histologic features of IBD, entire polyp, whereas polypoid dysplasia in IBD may whereas these features should be absent in the case of show a combination of adenomatous and nonadenoma- a sporadic adenoma. Although it has been proposed tous crypts. However, these observations cannot be used that polypoid dysplasia in IBD shows stronger expres- as definite criteria, as there is considerable morphologic sion of p53 and less frequent nuclear β-catenin labeling, overlap between sporadic adenomas in IBD and polypoid most practicing pathologists do not routinely use these (A) (B) FIGURE 11.13 This focus of dysplasia in a case of inflammatory bowel disease forms a distinct polyp that is elevated above the surrounding mucosa (A; high power view shown in inset). The surrounding mucosa shows chronic minimally active colitis with crypt distortion and increased basal lymphoplasmacytosis, consistent with the history of chronic idiopathic inflammatory bowel disease (B). 276 Neoplastic Gastrointestinal Pathology: An Illustrated Guide immunostains to distinguish between these lesions. The polyps are basally oriented with no significant hyper- distinction between a sporadic adenoma and polypoid chromasia, pseudostratification, nor abundant apoptotic low grade dysplasia in the setting of IBD may have lit- bodies. Neuroendocrine cells and mitotic figures may be tle clinical importance, because polypectomy appears present, but they are typically confined to the crypt bases. to be the adequate treatment in either case. The most The proliferative zone, which can be highlighted by Ki-67 important endoscopic question is whether the dysplasia immunohistochemistry, is expanded but limited to the is polypoid or not. If it is polypoid, polypectomy may be basal half of the crypt. Hyperplastic polyps can be mor- sufficient treatment, whereas a flat or indistinct elevated phologically subdivided into three types (microvesicular, area of dysplasia is more concerning and may require goblet-cell-rich, and mucin-poor), but it is not currently additional treatment in the setting of IBD. necessary to distinguish between them in clinical practice. SSA/P is a relatively recently described polyp that has Serrated Polyps distinct histologic and biologic features as compared to hyperplastic polyps. These polyps are common, compris- Serrated polyps are common overall, comprising up to ing 10% to 20% of serrated polyps. Prior to their relatively 15% to 45% of all colorectal polyps. The diagnostic clas- recent description, SSA/Ps were thought to be within the sification of serrated polyps has undergone significant spectrum of hyperplastic polyps, but it is now recognized change in the past two decades. The unifying histologic that SSA/Ps are precursor lesions to CRC via the serrated feature of these polyps is sawtooth or serrated architecture pathway. that is thought to occur due to alterations in both apop- SSA/Ps typically are flat and sessile endoscopically tosis and senescence of crypt epithelial cells. Although (Figure 11.15A–J), with indistinct borders and a mucin historically the majority of serrated polyps were classified cap. These polyps are usually greater than 5 mm in size, as hyperplastic polyps, it was observed that some cases of and often resemble a prominent colonic mucosal fold; SSA/ CRC appeared to arise in the context of certain serrated Ps are more common in the right colon than the left. The polyps, particularly those that were large and right-sided. defining histologic features of SSA/Ps are architectural, Based on distinct morphologic, genetic, and clinical find- rather than cytologic. The most distinct architectural ings, the WHO now classifies serrated polyps as follows: changes are identified at the base of the crypts, which show hyperplastic polyp, sessile serrated adenoma/polyp (SSA/P), abnormal branching and dilatation that can resemble a and traditional serrated adenoma (TSA) (see Table 11.3). “T,” “L,” “boot,” or fish mouth shape (Figure 11.15E–F). Hyperplastic polyps are the most common type of ser- The current minimum criteria for SSA/P consists of a sin- rated polyp, representing 75% to 90% of serrated lesions. gle dilated crypt with convincing architectural distortion. Endoscopically, hyperplastic polyps tend to be small (less In comparison to hyperplastic polyps, the serration of the than 5 mm), flat, pale, smooth, and located in the left glands in SSA/Ps extends to the base of the crypts, and is colon. These polyps have essentially no malignant poten- not confined to the upper half of the gland. Mature gastric tial. Histologically, hyperplastic polyps are characterized foveolar cells and goblet cells may also be found at the base by straight crypts that narrow at the base, with serration of the crypts, which is known as “reverse maturation.” An limited to the upper half of the crypts (Figure 11.14A–E). inverted growth pattern can occur when there is herniation The basement membrane beneath the surface may be thick- of crypts through the muscularis mucosae. Conventional ened. In comparison to SSA/Ps the crypt bases are not T- or cytologic dysplasia is not present in SSA/Ps although it L-shaped, nor are they dilated. The nuclei of hyperplastic may develop (see subsequent paragraphs). TABLE 11.3 Clinical, Endoscopic, and Molecular Features of Colorectal Serrated Polyps World Health Shape and Organization Endoscopic Most Common Malignant Molecular Classification Prevalence Appearance Colorectal Site Potential Features HP Very common Sessile/flat, smooth, Distal Minimal to none KRAS or BRAF mutation and pale SSA/P Common Sessile/flat mucous Proximal cap No dysplasia Present BRAF mutation and CIMP Dysplastic Significant MLH1 promoter hypermethylation TSA Rare Sessile or Distal Present KRAS mutation pedunculated BRAF mutation or CIMP Source: Modified from Lieberman DA, Rex DK, Winawer SJ, et al. Guidelines for colonoscopy surveillance after screening and polypectomy: a consensus update by the US Multi-Society Task Force on Colorectal Cancer. Gastroenterology. 2012;143:844–857. 11 Neoplasms of the Colon 277 (A) (B) (C) (D) (E) FIGURE 11.14 Hyperplastic polyps have straight crypts with epithelial serration that is most prominent in the upper half of the glands (A). A tangential section of the crypts shows a star-shaped configuration (B, arrow). The nuclei are basally oriented with no significant hyperchromasia, pseudostratification, or prominent apoptotic bodies. The basal zone of the crypts is narrow, without branching (C). A Ki-67 immunostain demonstrates that the proliferation zone of a hyperplastic polyp is confined to the basal crypts (D). This is in contrast to a tubular adenoma, in which the proliferation zone extends all the way up to the surface (E). 278 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 11.15 (continued) 11 Neoplasms of the Colon 279 (G) (H) (I) (J) FIGURE 11.15 Sessile serrated adenomas are flat and sessile, with indistinct borders (A, courtesy Dr. Rhonda Yantiss). Sessile serrated adenomas demonstrate prominently dilated crypts (B) as well as serrations that involve the entire length of the crypt (C–D). T- and L-shaped crypts are present at the base of the lesion (E [arrow], F). The base of SSA/P also contains mature goblet cells and foveolar-type cells, a feature known as “reverse maturation” (G). A Ki-67 immunostain demonstrates that the proliferation zone is at the sides of the crypts and extends to the upper region of the crypts, unlike tubular adenomas (See Figure 11.14E) (H). Sessile serrated adenomas can have associated perineurioma-like stroma (I), as well as underlying lipomatous change (J). SSA/Ps have an atypical proliferative area that can (Figure 11.15J). It remains unclear if this represents an be highlighted using Ki-67 immunostaining; staining can association between an SSA/P and a typical lipoma, or is be found in an irregular and asymmetric pattern any- a type of change induced in the mesenchymal tissue by the where from the base to the surface of the crypts. In addi- epithelium. tion, mitotic figures are “upwardly displaced” and located SSA/Ps that develop cytologic dysplasia (Figure above the base of the crypts, in contrast to the location of 11.16A–D) similar to that seen in conventional adeno- mitotic figures in hyperplastic polyps. This helps explain, matous polyps are termed “SSA/P with cytologic dys- in part, why the crypts may proliferate in a horizontal plasia.” Previously, these were often classified as mixed configuration leading to T- and L-shaped crypts. serrated/adenomatous polyps, or mixed hyperplastic/ Some SSA/Ps contain mesenchymal changes including adenomatous polyps. They

are now thought to represent perineuriomatous change (Figure 11.15I). Both the SSA/P molecular progression in SSA/Ps, and are clinically sig- and perineuriomatous areas have been reported to show nificant as they may progress to carcinoma more rapidly BRAF mutations. In addition, SSA/Ps have been associ- than conventional adenomas or SSA/P due to the addi- ated with lipoma-like adipose tissue in the submucosa tional molecular “hit.” Histologically, these lesions have 2 8 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 11.16 Sessile serrated adenomas with conventional-type dysplasia have a sharp transition to cytologic dysplasia that reaches the epithelial surface (A–B). The cytologic dysplasia is similar to conventional adenomas with elongated, hyperchromatic nuclei and pseudostratification. This sessile serrated adenoma with progression to high-grade dysplasia shows cribriform architecture (C) with residual serrated architecture in the background. The serrated form of epithelial dysplasia features prominent serration with dysplastic cells containing eosinophilic cytoplasm, more similar to a traditional serrated adenoma (D). areas characteristic of SSA/P, with an abrupt transition cells have basally or centrally located nuclei with striking to conventional-type cytologic dysplasia (adenomatous eosinophilic cytoplasm; given these features, these are change). The cytologic dysplasia is usually low grade, often referred to as “eosinophilic pencillate cells.” Unlike but high-grade conventional-type dysplasia can be seen conventional adenomas, mitotic figures are rare. In addi- as well. A “serrated” form of cytologic dysplasia has also tion, they show ectopic crypt buds in the villi that are been described, featuring more cuboidal cells with open oriented perpendicular to the long axis of the villi, and do chromatin, similar to TSA. not connect to the muscularis mucosae. This is in contrast TSAs are rare (1%–2% of serrated polyps), and to hyperplastic polyps and SSA/Ps in which the crypts almost exclusively located in the distal colorectum. are anchored to the muscularis mucosae. These ectopic Endoscopically, they may be pedunculated or sessile. crypts are the location of the proliferation zone and are Histologically, these polyps show a villous or tubulovil- one of the most characteristic histologic features of TSA; lous architecture and surface cytologic dysplasia (typi- however, they are reportedly only seen in approximately cally low grade) (Figure 11.17A–C). The tall and columnar one-third of cases. 11 Neoplasms of the Colon 2 81 (A) (B) (C) FIGURE 11.17 Traditional serrated adenomas have a complex villiform growth pattern at low power (A). Numerous ectopic crypts (B, arrows and C) are present along the sides of the villi. TSAs typically display low-grade cytologic dysplasia at the surface, with tall columnar epithelium with striking eosinophilic cytoplasm. Serrated polyposis syndrome is an ill-defined and prob- • Greater than 20 serrated polyps of any size found ably underrecognized disorder characterized by multiple anywhere in the colon. serrated polyps throughout the colorectum, with an associ- Based on molecular analysis, there are likely two sub- ated increased risk of CRC. The polyps can be hyperplastic types of this syndrome, with overlapping features. The polyps, SSA/Ps TSAs, or a combination thereof. The genetic first subgroup corresponds to the first criterion listed features of serrated polyposis syndrome are not well under- above (at least five serrated polyps, at least two greater stood. Compared to other syndromes with a predisposition than 10 mm). These polyps can be large hyperplastic pol- to CRC, this syndrome is unusual in that it primarily affects yps, SSA/P or SSA/P with dysplasia. These polyps com- older adults (although there is a wide age range). monly have molecular alterations including CpG island The criteria for diagnosing serrated polyposis syn- methylator phenotype (CIMP) and BRAF mutation; drome consist of at least one of the following: KRAS mutations are less common. The second subtype • At least five serrated polyps proximal to the sigmoid corresponds to the third criterion listed above (greater colon, two of which are greater than 10 mm. than 20 polyps of any size). The polyps in this subtype • Any number of serrated polyps proximal to the sig- are often hyperplastic polyps, and frequently have muta- moid colon in an individual who has a first-degree tions in KRAS. This subtype has a lower overall risk relative with serrated polyposis syndrome. of CRC. 2 8 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Molecular Alterations in Serrated Polyps 10 mm, the current guidelines recommend follow-up at 3 years, similar to follow-up recommendations for conven- KRAS mutations are more characteristically present in tional adenomas with high-risk features. SSA/Ps with dys- goblet-cell-rich hyperplastic polyps, while BRAF muta- plasia should be completely removed and patients should tions are associated with microvesicular hyperplastic undergo surveillance in 1 year. Patients with serrated pol- polyps, and some authors believe that a subset of microve- yposis syndrome should undergo surveillance every 1 to sicular hyperplastic polyps may progress to SSA/Ps. 3 years. These guidelines are summarized in Table 11.4. SSA/Ps are thought to develop through BRAF mutations and CpG island methylation. If CpG island methylation of the MLH1 promoter region occurs, the SSA/Ps may Diagnostic Challenges progress to dysplasia and possibly subsequent adenocarci- The most common diagnostic dilemma is distinguishing noma. The promoter hypermethylation causes downregu- between an SSA/P and another type of serrated polyp, lation of gene transcription with resultant decrease/loss of usually a hyperplastic polyp. Attention to the lack of dila- MLH1 protein expression; thus, MLH1 immunostaining tation, serration, and complex shapes at the base of the may be lost in areas of cytologic dysplasia. This molecular crypts typically helps support the diagnosis of hyperplastic alteration in the serrated pathway leads to microsatellite polyp rather than SSA/P. Poor orientation or tissue frag- instability and possibly to an accelerated path to adeno- mentation, however, may preclude a definite distinction carcinoma when additional molecular events occur. In between the two. If the distinction cannot be made due to fact, serrated precursor lesions are sometimes seen at the tissue fragmentation, lack of orientation, or the fact that edge of sporadic microsatellite unstable adenocarcinomas. a small biopsy of a large lesion is received for evaluation, TSAs can have KRAS or BRAF mutations or a CIMP. a diagnosis of “serrated polyp” can be rendered with an explanatory comment. Correlation with the macroscopic size and location of the polyp can also be very helpful. Treatment and Surveillance Guidelines A similar issue can arise when multiple fragments of ser- These guidelines are dictated by the risk of progression rated polyp are submitted, and some of them contain con- to malignancy. Most patients with hyperplastic polyps ventional-type cytologic dysplasia, as it may be difficult detected at initial colonoscopy should undergo repeat sur- to ascertain whether the tissue fragments represent SSA/P veillance after 10 years. More than four hyperplastic pol- and a separate conventional adenoma, or an SSA/P with yps at the time of endoscopy, and/or any hyperplastic polyp conventional dysplasia (progression). The identification of greater than 5 mm, may warrant follow-up at 5 years. SSA/ both the SSA/P area and conventional dysplastic portion Ps are followed with repeat endoscopy every 5 years, simi- in the same tissue fragment can help confirm this as pro- lar to conventional-type adenomas, and should be com- gression within the SSA/P. Furthermore, correlation with pletely removed if possible. When there are greater than the macroscopic findings (ie, whether one or two lesions three SSA/Ps at endoscopy or one SSA/P is greater than were sampled) can be very useful. TABLE 11.4 Recommended Surveillance Intervals After Endoscopic Resection of Polyps Histology Size Number Location Interval in Years Hyperplastic polyp (HP) <10 mm Any number Rectosigmoid 10 HP ≤5 mm ≤3 Proximal sigmoid 10 HP Any size ≥4 Proximal sigmoid 5 HP >5 mm ≥1 Proximal sigmoid 5 SSA/P or TSA <10 mm <3 Any site in colon 5 SSA/P or TSA ≥10 mm 1 Any site in colon 3 SSA/P or TSA <10 mm ≥3 Any site in colon 3 SSA/P ≥10 mm ≥2 Any site in colon 1–3 SSA/P w/dysplasia Any size Any number 1–3 TA < 10 mm 1–2 Any site in colon 5–10 TA <10 mm 3–10 Any site in colon 3 TA, TVA, VA Any size >10 Any site in colon <3 TA ≥10 mm Any number Any site in colon 3 VA or TVA Any size Any number Any site in colon 3 Adenoma with high-grade Any size Any number Any site in colon 3 dysplasia (HGD) Serrated polyposis syndrome Any size Any number Any site in colon 1 Source: Modified from Rex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of the colorectum: review and recommendations from an expert panel. Am J Gastroenterol. 2012;107:1315–1329; Lieberman DA, Rex DK, Winawer SJ, et al. Guidelines for colonoscopy surveillance after screening and polypectomy: a consensus update by the US Multi-Society Task Force on Colorectal Cancer. Gastroenterology. 2012;143:844–857. 11 Neoplasms of the Colon 2 8 3 TSA can also be in the differential diagnosis with SSA/P diamond-shaped crypts, and location in the distal colon although this is a less common dilemma. The villous archi- or rectum. Features favoring SSA/P include location in tecture, together with the low-grade surface dysplasia and the proximal colon and irregular T-shaped or fish-mouth- ectopic crypts, should help support the diagnosis of TSA. shaped crypts. Additionally, SSA/P typically lack promi- If a right-sided polyp has features suggestive of a TSA, nent inflammation, ulceration, granulation tissue, and the possibility of an SSA/P with progression and dyspla- fibromuscular proliferation in the stroma. sia should be considered. The most common polyp in the differential diagnosis of a TSA is tubulovillous adenoma. Tubulovillous adenomas typically show more significant COLORECTAL ADENOCARCINOMA dysplasia than that seen in TSAs, and do not contain ecto- pic crypts. Epidemiology Other entities in the differential diagnosis of SSA/P are prolapse and inflammatory-type polyps. These polyps CRC is the third most common malignancy in both women can contain elongation and serration of crypts that mimic and men, although men are more commonly affected. SSA/P (Figure 11.18A–C). Features favoring prolapse/ There is a great degree of geographical variation in the inflammatory-type polyps include inflammation, fibro- incidence of CRC, with industrialized nations having a muscular hyperplasia of the lamina propria, triangular or much higher incidence than developing countries due to (A) (B) (C) FIGURE 11.18 Prolapse polyps may contain irregularly shaped hyperplastic crypts that mimic SSA/Ps; however, the perpendicular fibromuscular stroma and inflammatory changes of prolapse polyps help to distinguish the two (A–C). 2 8 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide differences in diet as well as genetic alterations. Likewise, the gastrointestinal tract, CRC is usually composed of survival varies with geography, due to differences in the malignant glands (Figure 11.19A–E). The glands may be availability of screening colonoscopy and the variation in incompletely formed and have central dirty or comedo- the quality of medical care among different countries. type necrosis. The nuclei often have vesicular chromatin Both genetic and environmental factors influence the with prominent nucleoli. The glands are often angu- risk of development of CRC. Genetic polyposis syndromes lated and surrounded by a fibroinflammatory stromal (see also Chapter 6) have a strong penetrance, but account response. for only a minority of cases (approximately 5%). Some The most common immunohistochemical profile cases of CRC appear to be familial, but no known genetic of colorectal carcinoma is cytokeratin (CK)20 positive, mutation is detectable. Chronic idiopathic inflammatory CDX2 positive, and CK7 negative. However, not all bowel disease, which has a strong genetic component, is CRCs show this immunoprofile (see also Chapter 13). also a risk factor for CRC, particularly in the context of CK20 positivity is more common in well-differentiated long-standing disease. Table 11.5 summarizes diseases tumors than poorly differentiated ones. CK7 positivity, associated with increased risk of CRC. when present, is often patchy, and is more commonly One of the greatest risk factors for the development found in the rectum where approximately 25% of rec- of CRC is advanced age. Additional risk factors include a tal adenocarcinomas express of CK7. CDX2 expression prior history of CRC, an adenomatous polyp greater than is present in about 70% of CRC, and is more common 1.0 cm, a first-degree relative with a history of CRC (fam- in well- and moderately differentiated tumors compared ily history), African American ethnicity, smoking, alco- to poorly differentiated tumors. Microsatellite unsta- hol consumption, diabetes mellitus, physical inactivity, ble tumors are more commonly negative for CK20 and obesity, previous uretero-sigmoidectomy, and a history CDX2, and medullary carcinoma

is negative for CDX2 of abdominal irradiation. Conversely, environmental fac- in approximately 85% of cases. Signet ring cell carcinoma tors that are protective against CRC include diets rich in expresses CK7 in up to 33% of cases. fibers and vegetables, physical activity, nonsteroidal anti- The histologic classification of CRC from the WHO inflammatory drugs, and folate, among others. is summarized in Table 11.2. When a subtype of CRC is present but the percentage fails to meet the WHO diag- nostic criteria for classification as that subtype, the minor Clinical Features component(s) may be listed in the report along with the Patients who present with CRC often have vague general- diagnosis of conventional adenocarcinoma (eg, poorly ized complaints such as weight loss or fever. Symptoms differentiated adenocarcinoma with mucinous and signet often include changes in bowel habits, stool size, and ring cell components). hematochezia. Left-sided CRCs are more likely than right- Mucinous adenocarcinoma of the colorectum is sided CRCs to present with a change in bowel habits and diagnosed when greater than 50% of the tumor is com- hematochezia, due to the smaller lumen of the bowel on posed of mucin pools, with tumor cells floating within the left side. Rectal cancer can present with pain and tenes- the mucin (Figure 11.20A–B). The floating epithelium mus as well as pencil-thin stools. Alternatively, right-sided commonly appears as detached strips, and there may be CRCs are more likely to present with anemia rather than a minor component of signet ring cells. This subtype of obstruction or change in bowel habits. Laboratory find- CRC is relatively common, accounting for 10% of all ings can include iron deficiency anemia, elevated serum CRC. Mucinous adenocarcinoma is more common in carcinoembryonic antigen, and, rarely, Streptococcus microsatellite unstable tumors and younger patients. bovis bacteremia. The prognostic significance of this subtype has been disputed, but it appears that mucinous adenocarcino- Macroscopic and Microscopic Features mas associated with microsatellite instability have a more favorable course than those that are microsatellite Colorectal cancer can have several different growth pat- stable. Of note, the term “mucinous adenocarcinoma” terns macroscopically, including polypoid/exophytic, should not be used to describe the extracellular mucin ulcerating, and endophytic lesions. When CRC grows cir- pools found after neoadjuvant chemoradiation in rectal cumferentially around the lumen of the large bowel, it is cancer. described as annular. Rarely, CRC has a diffuse growth Signet ring cell carcinomas are the third most com- pattern with indistinct borders, similar to linitis plastica mon subtype of CRC, accounting for approximately 1% of of the stomach. CRC. Designation as a signet ring cell carcinoma requires Histologically, about 85% of CRC are intestinal- that greater than 50% of the tumor be composed of sig- type adenocarcinomas, with the remaining 15% com- net ring cells. Signet ring cells, characterized by a nucleus prising various subtypes that will be discussed in the that is eccentrically displaced by a mucin vacuole (Figure subsequent paragraphs. Similar to the remainder of 11.21A–B), are typically dyscohesive with an infiltrating 11 Neoplasms of the Colon 2 8 5 TABLE 11.5 Diseases Associated With Colorectal Adenocarcinoma Familial MUTYH- Juvenile Adenomatous Peutz-Jehgers Cowden Associated Polyposis Polyposis (FAP) Lynch Syndrome Syndrome Syndrome Polyposis Syndrome Ulcerative Colitis Crohn disease Inheritance mode Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Partial contribu- Partial contribution/ Dominant Dominant Dominant Dominant Recessive Dominant tion/penetrance penetrance of of multiple multiple genes genes Molecular APC (5q21–q22) MLH1 (3p21–p23), STK11/LKB1 PTEN MUTYH (former SMAD4/DPC4 Variable Variable alterations MSH2 or MSH6 (19p13.3) (10q23) MYH) (1p34.1) (18q21.1) or (2p21), PMS2 BMPR1A/ALK3 (7p22) (10q22.3) Premalignant Adenomas Adenomas Hamartomas with Hamartomas with Adenomas Juvenile polyp with Dysplasia Dysplasia lesions dysplasia dysplasia dysplasia Polyp number 100s–1,000s Varies Varies Varies 5–100s 3–200 Varies Varies Location of polyps Colorectum Colorectum Small intestine, Stomach, small Colorectum, Colorectum, Colorectum Colorectum within the GI tract (especially left (especially right stomach, intestine, small intestine, small intestine, colon), small colon), small colorectum colorectum stomach stomach intestine intestine Lifetime risk for 100%, mean age 10–53% 39% Little or no risk 93-fold increased 39%, Relative risk 30% risk by age Relative risk 2.9% at developing 35–40 risk 34 30 in patients 10 years following colorectal with pancolitis diagnosis adenocarcinoma before age 15 years Recommendations Lower & upper Colonoscopy Lower & upper Baseline Biannual lower Lower & upper Variable Variable for surveillance endoscopy every 1–2 years endoscopy colonoscopy & upper endoscopy every 1–5 years from age 20–25 every 2–3 years at age 35 years endoscopy from annually from depending on years or 10 from age 18 then every 5 age 25-30 years age 15 years polyp location years prior to years to 10 years or and burden the earliest age more frequently of colon cancer if symptomatic diagnosis in or polyps are the family noted (whichever comes first) Source: Data from Arber and Moshkowitz, Curr Gastroenterol Rep. 2011;13:435-41; Canavan et al, Aliment Pharmacol Ther. 2006;23:1097-104; Jass, Pathol Res Pract. 2008;204:431-47; Koornstra, Best Pract Res Clin Gastroenterol. 2012;26:359-68; Pan, World J Gastrointest Oncol. 2011;3:33-42; Rampertab et al, Gut. 2003;52:1211-4; Adapted from WHO classification of tumours of the digestive system, 4th edition, 2010. 2 8 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 11.19 Low-grade colorectal adenocarcinomas are composed of more than 50% glands (A–B). Dirty necrosis is a common feature. Poorly differentiated adenocarcinomas (high grade) have predominately solid and cribriform growth patterns with only focal gland formation, and high-grade nuclear features (C–D). Tumors with numerous intratumoral lymphocytes should raise the possibility of MSI and Lynch Syndrome (E). 11 Neoplasms of the Colon 2 87 (A) (B) FIGURE 11.20 Mucinous adenocarcinomas feature lakes of dissecting mucin containing floating strips of malignant epithelium (A–B). The epithelium typically has low-grade cytologic features (B). growth pattern. Signet ring cell tumors are usually aggres- negative for neuroendocrine markers. Medullary carcino- sive and are considered by convention to be poorly dif- mas may not express CK20 or CDX2, and occasionally ferentiated. This subtype has also been associated with express CK7 in contrast to the typical immunoprofile of microsatellite unstable CRC. CRC. Despite the aggressive appearance of medullary car- Medullary carcinoma is a unique subtype of CRC that cinoma, this tumor has a more favorable prognosis, as do is strongly associated with MSI. Microscopically, these other tumors associated with MSI. tumors are composed of uniform cells that may have a There are several rare subtypes of CRC. Pure squamous round or polygonal shape, with eosinophilic cytoplasm and cell carcinomas are composed of invasive nests of cells with a characteristic lymphocytic infiltrate (Figure 11.22A–B). variable keratin formation. Primary squamous cell carci- The tumor cells may form nests and cords, and therefore, noma of the colorectum is a diagnosis of exclusion, as prox- may mimic NETs. However, medullary carcinomas are imal extension of an anal tumor, metastatic disease, and (A) (B) FIGURE 11.21 Areas of signet ring cell adenocarcinoma (arrows) can occur along with a component of conventional intestinal-type adenocarcinoma (A). The signet ring cells are dyscohesive, have an eccentrically placed nucleus, and intracytoplasmic vacuoles that contain mucin (B). 2 8 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 11.22 This medullary carcinoma has an associated lymphocytic Crohn-like reaction at the periphery (arrows), typical of microsatellite unstable tumors (A). This tumor is composed of sheets of polygonal, monotonous cells with an intratumoral inflammatory infiltrate composed mainly of lymphocytes (B). squamous cell carcinoma arising from a fistula tract must Other rare variants include sarcomatoid carcinoma, all be excluded. These tumors tend to have a very poor prog- which is a biphasic tumor composed of both carcinoma- nosis. Clear cell adenocarcinoma of the colon is composed tous and sarcomatous components; choriocarcinoma, of polygonal cells arranged in nests (Figure 11.23A–B). The which usually demonstrates foci of conventional CRC as cytoplasm of the tumor cells is optically clear to eosino- well as areas of both cytotrophoblasts and syncytiotro- philic. There is often a conventional adenocarcinoma com- phoblasts; and adenosquamous carcinoma, which shows ponent and/or a tubular adenoma adjacent to this tumor, areas of both squamous cell carcinoma and adenocar- which, along with immunohistochemistry (CK20+ and cinoma and may be associated with hypercalcemia and CDX2+, negative for PAX8 and PAX2) helps to distinguish elevated levels of Parathyroid hormone (PTH)-related it from metastatic clear cell carcinoma of the kidney. protein. (A) (B) FIGURE 11.23 Clear cell carcinoma is a rare subtype of colorectal carcinoma that can occur in association with conventional intestinal-type adenocarcinoma (A). The cell borders are well-defined and contain clear cytoplasm (B). 11 Neoplasms of the Colon 2 8 9 Adenocarcinoma presenting in a polyp may lead to infiltrating small angulated glands or single cells, and unique diagnostic challenges. A malignant polyp is defined architectural complexity including a cribriform growth as one that harbors adenocarcinoma extending beyond pattern and/or solid sheets of cells (Figure 11.24A–B). the muscularis mucosae into the submucosa of the polyp. Furthermore, in true invasion, the malignant epithelium Carcinoma confined to a polypectomy specimen represents tends to be of a higher grade in the invasive component a unique circumstance in which the biopsy specimen may than in the overlying adenoma. Invasive adenocarcinomas be both diagnostic and curative. Depending on the micro- also typically lack associated hemorrhage and hemosid- scopic findings in the specimen, patients may not need to erin-laden macrophages seen in cases of misplaced ade- undergo further surgical treatment for their carcinoma. nomatous epithelium. If mucin pools are present in cases Malignant polyps that are pedunculated can often be of invasive adenocarcinoma, they characteristically have safely managed by endoscopic resection, provided pathol- irregular dissecting borders and contain malignant cells. ogists are able to evaluate specific histologic features. It is Histologic features that favor misplaced epithelium helpful for the polyp to be removed as a single piece with a include the presence of lamina propria around the mis- clearly defined stalk margin, whereas one may not be able placed glands, hemorrhage, hemosiderin-laden macro- to evaluate the margin status of fragmented specimens. phages, and rounded glands in an orderly arrangement Patients whose margin status is uncertain may need to (Figure 11.24C–E). Misplaced epithelium does not have undergo further resection, although sometimes rebiopsy associated desmoplasia, and does not contain angulated of the polypectomy site may prove sufficient if the rebi- glands. Misplaced epithelium can sometimes contain opsy is negative. In contrast, the extent of invasive carci- high-grade dysplasia and even architectural complexity. noma in sessile polyps may be difficult to appreciate, and However, the degree of cytologic atypia should be similar endoscopic resection may not be adequate. to the dysplasia in the surface adenomatous epithelium. Once the diagnosis of invasive adenocarcinoma is Benign mucin pools in the submucosa have round edges made in a polypectomy specimen, there are three critical and do not contain epithelial cells, although low-grade morphologic features that must be assessed and reported: epithelium at the peripheries of mucin pools may be pres- margin status, tumor grade, and lymphovascular invasion. ent. In addition, the epithelium associated with the benign An increased risk of adverse outcome has been associated mucin pools should be similar in grade to the mucosa in with tumor present less than 1 to 2 mm from the cauter- the overlying associated polyp. ized margin. The presence of lymphovascular invasion is Another diagnostic challenge in the diagnosis of CRC another histologic feature that is unfavorable. Retraction is the distinction from endometriosis, particularly when artifact around malignant glands can mimic lymphovas- endometriosis involves the wall of the colon. Endometriosis/ cular invasion, so if there is any doubt about the presence endosalpingiosis occurs in women of reproductive age, of lymphovascular invasion, immunostains such as D2-40 who often present with infertility, hematochezia (some- or CD31 may be helpful. Finally, the presence of any high- times coinciding with menstruation), or abdominal pain. grade (poorly differentiated) component of invasive car- Endoscopically, endometriosis may present as a polyp or cinoma is considered a risk factor for disease recurrence mass, or may be found incidentally. Endometriosis may or regional lymph node metastases. Thus, the presence of be found in the serosa, muscularis propria, submucosa, any poorly differentiated component in a malignant polyp or lamina propria. It is histologically composed of benign should be noted in the pathology report. Other histologic haphazardly arranged columnar glands reminiscent of features that some authors have found to correlate with endometrium (Figure 11.25A–C). The columnar epithelium adverse prognosis include the depth

of submucosal inva- may appear hyperchromatic, but does not contain goblet sion and tumor budding. Although these features have not cells. Endometrial stroma may be present, which can vary been incorporated into patient management guidelines in morphology depending on the menstrual cycle. Features yet, future studies may warrant their inclusion. favoring endometriosis include identification of endometrial stroma, benign nuclear features, a lack of “dirty” necrosis Diagnostic Challenges (seen in CRCs), and hemosiderin deposition. The endome- trial epithelium is positive for CK7 and PAX8, while nega- One major pitfall in the diagnosis of invasive adenocarci- tive for CK20 and CDX2, in contrast to typical CRC. In noma is misplaced adenomatous epithelium, also known addition, the endometrial stroma may be decidualized and as pseudoinvasion, particularly in pedunculated adeno- expresses CD10, ER, and PR. Rarely, Müllerian tumors mas of the distal colorectum. The primary mechanism may develop from endometriosis in the colon. for pseudoinvasion is thought to be traumatic mechanical Endosalpingiosis is thought to be due to metaplasia forces that push mucosa into the submucosa, similar to of the peritoneal mesothelial surface into an epithelial colitis cystica profunda. type that resembles fallopian tube epithelium. It is usually Microscopic features favoring invasive adenocar- an incidental finding. Because it is due to metaplasia of cinoma include a prominent desmoplastic response, mesothelial cells, it is commonly found on the peritoneal 2 9 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 11.24 This conventional adenoma contains invasive adenocarcinoma; note the complex cribriformed architecture (A). A surrounding desmoplastic reaction and high grade nuclear features are readily apparent (B). This adenoma contains misplaced epithelium deep to the surface; the degree of dysplasia in the misplaced epithelium is similar to that seen at the surface (C). Other features favoring misplaced glands over invasive adenocarcinoma include surrounding lamina propria and hemosiderin-laden macrophages (D). The mucin beneath the surface of an adenoma with prolapse-type changes shows rounded edges that do not dissect tissue (E). 11 Neoplasms of the Colon 2 91 (A) (B) (C) (D) FIGURE 11.25 Endometriosis of the bowel may mimic invasive adenocarcinoma, and may even form a mass (A). However, the columnar epithelium lacks goblet cells, and there is associated endometrial stroma, hemosiderin-laden macrophages, and hemorrhage (A, arrow, and B). High-grade nuclear features should be absent as well, although nuclei may be enlarged and hyperchromatic (C). Endosalpingiosis is commonly found on the peritoneal surface of the bowel, and may also mimic adenocarcinoma (D). Histologically, the epithelium is very low grade, and there is no associated desmoplastic reaction to the glands. surface (Figure 11.25D). Histologically, endosalpingiosis contains many changes and clarifications from previous is composed of tubular-type epithelium that is ciliated, editions. Fortunately, many of the pathologic parameters and endometrial-type stroma is not present. The dis- are relatively straightforward, but some of the staging tinction of endosalpingiosis from CRC is usually more parameters remain challenging and/or controversial and straightforward than endometriosis, but if the diagnosis require additional explanation. These include the concept is not considered it can be missed. Features favoring endo- of intramucosal adenocarcinoma, the identification of salpingiosis include primarily peritoneal location, low- invasion through the muscularis propria and into perico- grade cytologic features, and ciliation. lonic/perirectal soft tissues (T3), proper classification of serosal involvement (T4a) or adhesions to adjacent organs Grading and Staging (T4b), evaluation of total mesorectal excision (TME), assessment of acellular mucin pools after neoadjuvant CRCs are graded and staged according to the seventh chemoradiation, tumor regression grade, tumor budding, edition of AJCC TNM, which was released in 2009 and and the definition of tumor deposits and N1c. 2 9 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Similar to other adenocarcinomas, CRCs have tradi- T3 tionally been graded as well, moderately, and poorly dif- The identification of invasion through the muscularis pro- ferentiated; the justification for this is that the degree of pria and into pericolonic soft tissue (pT3) can be challeng- differentiation is an independent predictor of outcome. ing when invasion by the leading edge of tumor is obscured Due to interobserver variability in the distinction/inter- by a fibrotic reaction to the tumor, which makes it diffi- pretation of well-differentiated from moderately differen- cult to determine where the muscularis propria ends and tiated tumors, however, a two-tiered system is currently the pericolonic adipose tissue begins. This can be particu- used. Tumors are categorized as either low grade (well- larly challenging if invasion into the pericolonic soft tissue and moderately differentiated) or high-grade (poorly is focal. It has been proposed that the loss of muscle fibers differentiated). Tumors exhibiting less than 50% gland between glands of tumor at the invasive front should be formation are classified as high grade when assessing the classified as pT3, but long-term studies are necessary to entire area of the tumor. determine if these “minimal” pT3 cases behave similarly According to the American Joint Committee on to those that are unequivocally classified as pT3. Taking Cancer (AJCC), seventh edition, carcinoma in situ and additional sections of the area in question may also be tumors for which invasion is restricted to the lamina helpful in getting a better look at the interface between the propria are staged as carcinoma in situ (Tis), whereas muscular wall and the pericolonic soft tissue. tumors that invade the submucosa are staged as T1. Once a tumor has invaded the muscularis propria, it is staged as T2. Invasion of the pericolorectal soft tissues beyond T4a the muscularis propria is considered T3. When a tumor Once a tumor has invaded beyond the muscularis propria is present at the visceral peritoneal surface, it is staged as and into the pericolorectal soft tissues, determination of T4a. Finally, invasion or adherence of adjacent organs is whether the serosal surface is involved is of vital impor- considered T4b. In most circumstances, analysis of the tance. A tumor is classified as pT4a when it is present at depth of invasion is straightforward. However, certain the visceral peritoneal surface or the tumor has perforated scenarios can present diagnostic challenges that can lead the bowel wall (Figure 11.26A–B). Assessment of serosal to inconsistent or incorrect staging; these are discussed in involvement is important because it may indicate the need the immediately following sections. for systemic adjuvant chemotherapy, even in those patients with stage II (lymph node negative) cancers (most of whom would not receive adjuvant chemotherapy unless there are Tis and T1 high-risk features such as inadequate evaluation of the As mentioned in the previous paragraph, most patholo- lymph nodes, T4 pathologic stage, lymphovascular inva- gists consider carcinoma in situ to be within the spec- sion, bowel perforation, or poorly differentiated tumor). trum of high-grade dysplasia in the colon, and the use One important study regarding the importance of sero- of this term is discouraged because this lesion is not sal surface involvement was conducted by Shepherd et al., believed to have the potential to metastasize. In the in an analysis of 412 colon cancers in which local perito- AJCC, seventh edition, however, the term carcinoma neal involvement (LPI) was carefully assessed. Tumors that in situ (Tis) is included, and (unlike most other organs) had spread beyond the muscularis propria were classified the definition includes tumors that are restricted to the into four groups: tumors cells well clear of the peritoneal glandular basement membrane as well as tumors that surface (LPI1), tumor cells close to but not at the serosal invade the lamina propria (sometimes called intramu- surface with an associated mesothelial inflammatory and/ cosal carcinoma). The logic for this classification of or mesothelial hyperplastic reaction (LPI2), tumor cells Tis rather than T1 for intramucosal carcinoma in the present at the serosal surface with an inflammatory reac- colon arose from studies by Fenoglio et al. that demon- tion, mesothelial hyperplasia, or “ulceration” (LPI3), and strated the absence of lymphatics in the lamina propria free floating tumor cells in the peritoneum with evidence of the large bowel. It was therefore hypothesized that of adjacent “ulceration” (LPI4). LPI groups correlated with carcinoma that only invaded the lamina propria, but not overall survival, predicted intraperitoneal recurrence, and the submucosa, did not have the ability to metastasize. were a stronger independent prognostic indicator than the Whether or not the lamina propria of the bowel contains extent of local spread and lymph node involvement. lymphatics remains controversial, but most authorities Controversy remains, however, in cases where tumor believe that colorectal tumors confined to the lamina cells are close (less than 1 mm) to the serosal surface propria have minimal, if any, chance of metastasis. with circumstantial evidence of serosal involvement such Although the classification of Tis exists for the colorec- as hemorrhage, ulceration, and/or a mesothelial reac- tum in the AJCC scheme, many authorities discourage tion (Figure 11.26C). Colorectal tumors less than 1 mm the use of this term. from the serosal surface with a fibroinflammatory tissue 11 Neoplasms of the Colon 2 9 3 (A) (B) (C) FIGURE 11.26 This case of colorectal carcinoma shows malignant glands present at the inked serosal surface and is staged as T4a (A). A different case shows necrosis extending from the tumor to the serosal surface; this would be considered T4a as well (B). This case shows tumor that is less than 1.0 mm from the serosal surface, with a fibrous reaction (C); staging as T3 versus T4a is controversial. Many would stage as T3 with a comment in the report. reaction have been shown to have positive serosal scrape challenges in the interpretation and reporting of elastic cytology and outcome more similar to T4 tumors, thus lamina invasion. The elastic lamina is not easily identifi- suggesting peritoneal involvement. In cases where serosal able in all colon cancer cases, notably the right colon. In involvement is considered, it is often helpful to get deeper addition, the subserosal elastic lamina often does not form levels and take additional samples of the serosal surface. a contiguous line. In our own practice, we have found that Tumor cells may involve natural clefts of the serosal sur- most cases do not show continuous staining and some face, and these areas should be carefully assessed and con- do not stain at all. Currently, it remains unclear how to sidered T4a if involved by tumor. Since identification of report tumor invasion of the elastic lamina and how this mesothelial cells can be challenging, some have advocated should affect staging, and studies examining the behavior the use of CK7 or calretinin to identify mesothelial cells, of tumors that show penetration of the elastic lamina, but but this has not proven to be useful due to the lack of not the visceral peritoneum, have shown mixed results. mesothelial cells lining the serosal surface in many cases. Therefore, reporting of elastic lamina invasion is not rec- Other authors have suggested that invasion of the sub- ommended, nor is routine use of the elastic stain to help serosal elastic lamina can be used as a surrogate marker stage or predict outcome. for serosal invasion. In the colon, the subserosal elas- A common misunderstanding in staging rectal tumors tic lamina is found near the peritoneal surface (Figure is the belief they cannot invade the serosal surface. 11.27A–D), and thus the hypothesis is that tumors that Proximal rectal tumors at or above the peritoneal reflec- have invaded through this subserosal elastic lamina are tion do have a serosal surface, and tumor involvement of more likely to gain access to the serosal surface and this surface would be classified as T4a. In addition, the behave more aggressively. However, there are several anterior serosa extends more distally than the posterior 2 9 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 11.27 This elastic stain image shows an intact elastic lamina (A, arrow) without invasion by tumor (A–B, high power). Note that the elastic lamina forms a nearly continuous line that is black on elastic stain. Another case shows tumor cells very close to the serosal surface (C, arrow). When this area of tumor was stained for elastin, carcinoma is present on both the mucosal and serosal sides of the elastic lamina (D, arrows), suggesting that the tumor may have gained access to the serosal surface. serosa in the rectum. Rectal tumors distal to the perito- Previous data suggested that tumors with serosal involve- neal reflection do

not have a serosal surface, however. ment behave worse than those with invasion of adja- Therefore, a case with tumor cells present at the peripheral cent organs. Therefore, the previous edition of AJCC edge/surface of a distal rectal specimen would be classified (sixth edition) classified serosal involvement as T4b and as a positive radial margin but not serosal involvement. involvement of adjacent organs as T4a. More recent data (Surveillance, Epidemiology, and End Results [SEER]) Invasion of Adjacent Organs (T4b) demonstrated that invasion of adjacent organs behaves worse than serosal involvement, which resulted in the cur- Invasion of adjacent organs, which is classified as T4b, is rent classification in the AJCC, seventh edition. usually straightforward. Adhesions between the resected colorectum and adjacent organs should be staged as T4b when tumor cells are present within the adhesion, or Radial (Circumferential) Margin directly invading adjacent organs. In other words, one and Mesenteric Margin need not identify carcinoma in an adjacent organ to assign There is a great deal of confusion inherent in the definitions a stage pT4b; tumor in the adhesion alone is adequate. of the radial (circumferential) margin and the mesenteric 11 Neoplasms of the Colon 2 9 5 margin. It is important to remember, as well, that these none extending to the muscularis propria. One should not definitions differ according to site in the colorectum. A be able to visualize the muscularis propria in a nearly com- radial (circumferential) margin is surgically created by plete specimen. The mesorectum is considered incomplete dissection of soft tissue. Thus, peritonealized surfaces when there is little bulk to the mesorectum, and/or defects (eg, the cecum) are not considered to be circumferential are present in the mesorectum that clearly communicate (radial) margins because they are not a surgically created with the muscularis propria. Assessment of complete- plane. Therefore, in a segment of colon encased by peri- ness can be particularly challenging in abdominoperineal toneum, only the surgically created mesenteric margin is resection specimens, because the area in which the distal equivalent to the radial margin. The remaining peritoneal- rectum crosses the levator muscles appears tapered, and ized surface is not a margin. The ascending and descend- this can be misinterpreted as distal coning. Robotically ing colons lack posterior serosalized surfaces; thus this performed resections are becoming more common as area is considered the circumferential/radial margin. Very well, and these types of specimens can also present chal- few pathologists (<10%) actually document the status of lenges if the specimen is damaged during robotic removal. the posterior radial margins of ascending and descending Controversy exists as to whether robotic procedures are colon cancers. Fortunately, available data suggest that this superior to, equivalent, or inferior to open resections; margin is likely of limited practical importance; tumors however, experienced surgeons are able to perform robotic that involve this margin are generally of advanced with TME with complete preservation of the mesorectum. regional lymph node or distant metastases. In the distal portion of the rectum not encased in Lymph Node Assessment peritoneum, the entire circumference of the bowel is con- All lymph nodes that are identified during gross evalu- sidered a radial margin. The more proximal rectum does ation of a colectomy should be submitted for histologic contain a peritonealized surface and, therefore, could evaluation. In the vast majority of cases, standard gross be staged as T4a when involved by tumor, while the dis- examination is sufficient to identify lymph nodes. Fat tal rectum would be staged as T3 with a positive radial clearing agents can help identify lymph nodes when stan- margin. The radial margin is considered positive if the dard grossing fails to yield sufficient numbers of lymph tumor is 1 mm or less from the nonperitonealized surface. nodes. These agents are composed of formalin, ethanol, Furthermore, if the tumor is present within a lymph node and acetic acid, and make lymph nodes appear firm and 1 mm or less from the nonperitonealized surface, this is white after fixation. However, routine use of fat clearing also considered a positive radial margin. A positive radial agents is not considered the standard of care and is not margin is predictive of both local recurrence and death in required for adequate lymph node dissection. rectal carcinomas. The typical minimum number of lymph nodes for adequate assessment of CRC ranges from 12 (College of Total Mesorectal Excision American Pathologists [CAP]) or 10 to 14 (AJCC, seventh Rectal cancers that lie below the peritoneal reflection are edition). It is important to note that when less than the often removed by a surgical procedure known as total minimal number of lymph nodes is identified, the desig- mesorectal excision (TME). This procedure is now con- nation Nx should not be used, but the tumor should be sidered the standard of care for low rectal cancers. The staged according to the number of nodes that are involved. adequacy of the resection, is a strong independent predic- It is well known that rectal tumors treated with neoad- tor of local recurrence of the tumor and is noted to be juvant chemoradiation tend to have fewer lymph nodes complete, nearly complete or incomplete depending on the identified, and lymphoid tissue may decrease with age as appearance of the mesorectal envelope (Figure 11.28). An well. Therefore, there is no suggested minimum number incomplete mesorectum may be a surrogate marker for the of lymph nodes for rectal cancers treated with neoadju- local biologic aggressiveness of the tumor, or may be due vant chemoradiation, but as many as possible should be to technical difficulties during the procedure. identified. Mesorectal resection specimens should be assessed The definition, nomenclature, and role of tumor nod- grossly in the fresh state, and the entire mesorectal surface ules or deposits in staging CRC have evolved with recent should be carefully examined; the worst appearing area iterations of the AJCC. The fifth edition AJCC (1997) should be used to score the specimen (Figure 11.28A–E). defined tumor deposits as foci of tumor measuring less The mesorectum is considered complete when it is bulky than 3 mm without residual lymph node architecture. In with a smooth surface or only minor surface irregulari- the sixth edition AJCC (2002), the contour, rather than ties. There should not be any defect greater than 5 mm the size, of the tumor focus was used to distinguish totally in depth, and there should not be coning (ie, tapering of replaced lymph nodes (rounded) from tumor deposits the distal aspect of the specimen). The mesorectum is con- (irregular contour), and the latter were then coded as V1 sidered nearly complete when there is moderate bulk to (microscopic venous invasion) or V2 (grossly evident dis- the mesorectum and only mild surface irregularities with ease), depending on microscopic/gross findings. Tumor 2 9 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) FIGURE 11.28 This example of a complete total mesorectal excision shows a bulky surface with no significant surface defects (A). A nearly complete total mesorectal excision has moderate bulk, with a small defect that does not extend to the muscularis propria (B, arrow). Incomplete total mesorectal excisions have defects (arrow) that extend to the muscularis propria (C–E). The narrow distal portion of the excision at the right of the specimen (D, abdominoperineal resection) should not be considered coning, and is due to the normal anatomical resection at the levators. 11 Neoplasms of the Colon 2 97 nodules helped define the T stage in both the AJCC, fifth In challenging cases, helpful features in distinguishing and sixth editions, and were considered a discontinuous lymph nodes from tumor deposits include round shape, extension of the primary tumor; the T stage was therefore peripheral lymphocyte rim, peripheral lymphoid follicles, upstaged when necessary. In contrast, tumor deposits do possible subcapsular sinus, residual lymph node pres- not count toward the T stage in the AJCC, seventh edition. ent in surrounding fibroadipose tissue, and a thick cap- Pericolonic tumor deposits are currently defined in sule (Figure 11.29A–C). When a tumor deposit is present the AJCC 7th edition as “discrete foci of tumor found within a discernible blood vessel, this should be recorded in the pericolic or perirectal fat or in adjacent mesentery as lymphovascular invasion rather than a tumor deposit. (mesocolic fat) away from the leading edge of the tumor Table 11.6 shows the evolution of tumor deposit defini- and showing no evidence of residual lymph node tissue.” tions from AJCC, 5th, 6th, and 7th editions. This definition relies on the pathologist’s interpretation of The AJCC 7th edition also created a new category what constitutes residual lymph node tissue, and in some termed N1c for cases in which there are tumor deposits cases this is subjective. Interobserver variability exists present, but no positive lymph nodes. The letter “c” was when defining tumor deposits, even among pathologists chosen since it was the next alphabetic option after N1a with an interest/expertise in gastrointestinal pathology. and N1b. N1c does not, by definition, indicate a worse (A) (B) (C) FIGURE 11.29 This photomicrograph shows an obvious lymph node containing metastatic adenocarcinoma; the residual peripheral lymphoid tissue is easily seen (A). This image shows focus of tumor with irregular borders and no definite residual lymph node architecture; this would be classified as a tumor deposit (B). This focus of metastatic carcinoma has a round shape and a thick capsule, which many would classify as a positive lymph node rather than a tumor deposit even though there is no residual lymph node tissue (C). 2 9 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 11.6 Evolution of Colorectal Carcinoma Tumor Deposits With Recent Editions of the American Joint Committee on Cancer Staging Manual Tumor Deposit AJCC Tumor Deposit Shape Other Definitional Edition Size Criteria Criteria Criteria Effect on T Stage Effect on N Stage Fifth Less than 3 mm No No residual lymph node Considered discontinuous None architecture tumor extension, upstage when needed Sixth No Irregular shape No residual lymph node Considered discontinuous None architecture tumor extension, upstage when needed Seventh No No No residual lymph node None N1c category used when architecture no other lymph node metastasis is present prognosis than N1a and N1b. In cases in which there are lacking metastatic lymph node disease, and may influ- both lymph nodes and tumor deposits involved, N should ence the use of systemic therapy in certain circumstances. be classified only by the number of positive lymph nodes Morphologically, lymphovascular invasion is identified (ie, N1a, N1b, N2a, N2b) and it is not appropriate to use as tumor cells within flat endothelial-lined spaces (Figure the N1c classification in this circumstance. Tumor depos- 11.30A). It is usually readily apparent on hematoxylin and its do not count toward the total lymph node count, but eosin (H&E) stained sections, although the use of antibod- the number of tumor deposits should be recorded indepen- ies such as D2-40, CD31, or elastin may be helpful in cases dently of the lymph node count per CAP protocol. where vessels and lymphatic spaces are difficult to identify with certainty. The presence of perineural invasion should also be dutifully sought in resection specimens, as it is an Selected Stage-Independent Issues independent prognostic factor as well (Figure 11.30B). The term lymphovascular invasion was created due to Tumor budding is a more recently described feature of the difficulty in distinguishing tumor invasion of small colorectal carcinoma that may be associated with aggres- lymphatic spaces from invasion of postcapillary venules. sive behavior. It is believed to represent a type of epithe- Regardless, the presence of lymphovascular inva- lial-to-mesenchymal transition, indicating that tumor cells sion is an independent predictor of outcome in patients have gained migratory capability, including loss of cell (A) (B) FIGURE 11.30 This example of lymphovascular invasion shows tumor cells present within endothelial-lined spaces (A). Perineural invasion consists of tumor cells within the perineurium of the nerve (B, arrow). 11 Neoplasms of the Colon 2 9 9 Assessment of tumor budding may be most useful in cases where treatment could be affected, such as when car- cinoma is confined to a polyp and additional resection is being considered, or in stage II colorectal cancers (lymph node negative), where a decision regarding adjuvant chemo- therapy could be affected by the amount of tumor budding. To date, however, tumor budding is not routinely reported in either polypectomies

or resections for CRC. Furthermore, although tumor budding has been independently associated with poor outcome and lymph node metastasis, large pro- spective studies assessing the long-term prognostic utility of this histologic parameter are still lacking. Neoadjuvant Chemoradiation The standard of care for locally advanced rectal cancers FIGURE 11.31 Tumor budding consists of groups of five (tumors that are T3 or T4 and/or lymph node positive) is neo- cells or less at the invasive front of the tumor. adjuvant chemoradiation followed by resection. Common histologic findings described after neoadjuvant chemother- apy include acellular mucin pools, fibrosis, necrosis, hemo- siderin deposition, foamy macrophages, and calcification. adhesion and loss of cell polarity. This gain of migratory Acellular mucin pools can be found in the wall of the rectum capability and loss of cell adhesion is believed to be one of as well as in lymph nodes (Figure 11.32A–B). Although it is the first biologic steps toward metastasis. Histologically, presumed that these pools once contained viable tumor cells, tumor budding is identified at the invasive front of the residual acellular mucin after neoadjuvant therapy pools tumor, and consists of groups of up to five cells (cords or is not used in staging, and only viable tumor cells within aggregates) (Figure 11.31) that “bud” or invade away from mucin pools count toward both T and N stages. Deeper lev- the invasive front. There are several scoring systems that els of blocks may be of help in this scenario. Although the have been developed for assessing the amount of tumor finding of acellular mucin pools is not used in staging, it is budding in any given case, but a standardized scoring sys- nonetheless useful to mention the presence of these pools tem has not been agreed upon. and their location within the surgical pathology report. (A) (B) FIGURE 11.32 After neoadjuvant chemotherapy, this tumor has acellular mucin pools present in the muscularis propria, but the only viable tumor cells are present in the submucosa (arrow), and thus it is classified as ypT1 (A). This lymph node shows acellular mucin pools with no residual tumor cells (B). 3 0 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide The tumor’s response to the chemoradiation is mea- should be used, indicating neoadjuvant therapy. Although sured as the tumor regression grade (Figure 11.33A–D). the clinical significance of tumor regression grading is not Different classification schemes exist to evaluate the tumor well understood, complete response of a colorectal tumor regression grade; AJCC 7th edition uses a score from 0 to to neoadjuvant chemotherapy is associated with improved 3. Complete response (score 0) is defined by the absence disease-free survival, decreased chance of recurrence, of viable cancer cells. Moderate response (score 1) is char- and decreased metastasis. Careful gross sampling of the acterized by rare residual single cells or small groups of tumor site should be performed to adequately assess if any cancer cells, while minimal response (score 2) includes viable tumor remains, especially if no visible gross lesion residual cancer outgrown by fibrosis. Poor response (score remains. 3) is defined by minimal or no tumor kill with extensive residual cancer. Not surprisingly, the grading of response to therapy shows considerable interobserver variability MOLECULAR GENETICS in several studies. The tumor regression grade should be OF COLORECTAL CANCER scored in the primary colorectal tumor and not in lymph nodes or other sites harboring metastatic tumor. When The molecular basis of CRC is one of the best studied these cases are pathologically staged, the prefix “y” among human neoplasms, in large part because adenomas (A) (B) (C) (D) FIGURE 11.33 Tumor status post neoadjuvant therapy with no identifiable tumor cells, but only acellular mucin pools and fibrosis, is graded as tumor regression grade 0 (A). Tumor regression grade 1 shows rare malignant glands in mucin pools (B, arrows). Tumor regression grade 2 has foci of tumor, but with a greater proportion of fibrosis (C). Tumor regression grade 3 shows no definite tumor response to therapy (D). 11 Neoplasms of the Colon 3 01 are easily accessible via colonoscopy. It has long been with classic “carcinoid syndrome” symptoms such as diar- known that CRC usually develops from adenomatous pol- rhea, cutaneous flushing, bronchospasms, and venous tel- yps, with an interval of approximately 10 to 15 years for angiectasia is quite rare among tumors at this site. NETs the development of invasive carcinoma from an adenoma. arising in the ascending and transverse colon are more Fearon and Vogelstein expanded on Knudson’s two-hit likely to have metastatic potential, which correlates with hypothesis model of tumor suppressor genes and proposed size, presence of lymphovascular invasion, grade, and stage. a model of colorectal tumorigenesis with four features: Most rectal NETs measure less than 1 cm. 1. CRC has both activating mutations in oncogenes and Macroscopically, these tumors often appear as submuco- inactivating mutations in tumor suppressor genes. sal nodules (Figure 11.34A–D); larger tumors may pro- 2. At least four to five genes must be mutated for CRC duce overlying mucosal ulceration. Right-sided NETs are to arise. frequently larger than NETs arising in the distal colon and 3. The accumulation of genetic changes, rather than the rectum. Similar to NETs elsewhere in the gastrointestinal chronologic order of those changes, determines the tract, these tumors are gray in color when fresh and turn biologic behavior of CRC. yellow after fixation in formalin. A comprehensive discus- 4. Tumor suppressor genes with mutational inactivation sion of the histology, immunophenotype, and grading of of only one allele still appear to exert a phenotypic NET/NEC is found in Chapter 3. effect on the tumor. Diagnostic Challenges In addition to alterations of genes by mutations, dele- tions, and insertions, epigenetic mechanisms can influence The distinction between CRCs and NETs is generally the expression of genes in CRC. These epigenetic mecha- straightforward by routine histology. The acinar pattern nisms include cytosine methylation of DNA, histone mod- of low-grade NETs occasionally mimics a well-differen- ification, and microRNAs, among others. tiated adenocarcinoma, but neuroendocrine markers will Currently, three separate but overlapping pathways have confirm the distinction. It is important to remember that been identified for the development of CRC: the chromo- MOC31 is not useful in this situation, as it will mark both somal instability pathway, the CIMP pathway, and the mic- types of tumors. The solid growth pattern of NETs may rosatellite instability/mismatch repair pathway. These are occasionally resemble a low-grade lymphoma; this differ- discussed and illustrated in detail in Chapters 6, 13, and 14. ential can also be resolved with immunohistochemistry. Prostate cancer can sometimes enter into the differential diagnosis for rectal NETs, given the location and low- grade cytologic findings. Immunohistochemistry for pros- NEUROENDOCRINE NEOPLASMS tate markers (PSA and PSAP) as well as neuroendocrine markers help make this distinction. When seen in the liver, Epidemiology and Clinicopathologic Features the trabecular pattern of NETs may mimic hepatocellular Well-differentiated neuroendocrine tumors (NETs), for- carcinoma. Hepatocellular markers such as HepPar1 do merly known as carcinoid tumors in the tubular gut, not typically mark NETs, and neuroendocrine markers are now referred to as NETs in the current WHO 2010 do not typically stain hepatocellular carcinomas (with the Classification. NETs of the colon and rectum account for exception of the fibrolamellar variant, which can coex- 11% and 20% of all gastrointestinal tumors, respectively press synaptophysin and some hepatocellular markers). (see also the detailed discussion in Chapter 3). The most Mixed adenoneuroendocrine carcinomas (MANECs) common location for NET in the large bowel is the rec- are often a diagnostic consideration in high-grade NECs tum, followed by the cecum. Most colonic NETs arise of the colon and rectum. This distinction is further com- sporadically; in contrast to NETs elsewhere in the gastro- plicated by the fact that NECs usually arise from colorec- intestinal tract, there is no association with multiple endo- tal adenomas and adenocarcinomas. The diagnosis of crine neoplasia type 1 syndrome. MANEC requires a neuroendocrine component of at least Poorly differentiated neuroendocrine neoplasms are 30%, which should be confirmed by immunohistochemis- now referred to as neuroendocrine carcinomas (NECs) try. Approximately 40% of CRCs will have at least focal in the WHO 2010 Classification. The term NEC encom- staining with neuroendocrine markers, and this does not passes small cell and large cell neuroendocrine carcinoma fulfill the criteria for MANEC. Staining for neuroendo- (as well as mixed small and large cell carcinomas). NECs crine markers is not recommended unless neuroendocrine can arise anywhere in the colorectum, and are very aggres- histologic features are present. sive tumors regardless of location or morphologic subtype. The staging of NETs of the colon and rectum is slightly Rectal NETs are typically asymptomatic, and are inci- different than adenocarcinomas from the same location, dentally found at the time of colonoscopy for another rea- and Table 11.7 highlights these differences. The staging son. They are characteristically small, and tend to behave in guidelines for adenocarcinomas of the colon and rectum a benign fashion with low metastatic potential. Presentation should be used for NECs as well as MANECs. 3 0 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 11.34 This well-differentiated neuroendocrine tumor forms a submucosal mass with a yellow cut surface (A). Well-differentiated neuroendocrine tumors are composed of nests and trabeculae of monotonous cells with amphophilic cytoplasm and “salt-and-pepper” chromatin (B). This poorly differentiated neuroendocrine carcinoma shows high-grade cytologic features with nuclear pleomorphism and large nucleoli (C, D). MESENCHYMAL TUMORS comparison, leiomyomas of the muscularis propria (intra- mural) and leiomyosarcomas are very rare in this location, A detailed discussion of mesenchymal tumors (including and are more likely to present with clinically evident rectal gastrointestinal stromal tumor [GIST] and mesenteric bleeding or obstruction. fibromatosis) is given in Chapter 5. This section will focus in brief on the most common mesenchymal tumors of the Lipomatous Tumors colon and rectum. Lipomas are the most common submucosal mesenchymal polyp in the colorectum; they are rarely seen in the mus- Smooth Muscle Tumors cularis propria or subserosa as well. They are more often Leiomyomas of the colon most often arise from the found on the right side of the colon and in older women. muscularis mucosae, and are similar to smooth muscle Small lipomas (less than 2 cm) are usually asymptom- tumors found elsewhere in the body (Figure 11.35A–B). atic, while larger lesions (greater than 2 cm) may present They often present as polyps in the distal colon, and are with abdominal pain, bleeding per rectum, or a change in usually found incidentally during screening colonoscopy. bowel habits. Lipomas are composed of mature adipocytes They are slightly more common in men than women. In typically based in the submucosa (Figure 11.36A–B). The 11 Neoplasms of the Colon 3 0 3 TABLE 11.7 Comparison of Staging Criteria for Colorectal Neural Lesions Adenocarcinoma and Colorectal Neuroendocrine Tumor Ganglioneuromas (GNs) (Figure 11.37A) are most often Colorectal Adenocarcinoma Colorectal found in the colon as solitary sporadic lesions. These are and Poorly Differentiated Well-Differentiated usually incidental findings on the left side of the colon, Neuroendocrine Carcinoma Neuroendocrine Tumor measuring less than 1.0 cm. GNs are also found in the context of ganglioneuromatous polyposis, which is associ- pTis Carcinoma in situ, intraepithe- — lial (no invasion of lamina ated with Cowden syndrome and NF1, and in the context propria) of diffuse ganglioneuromatosis, which is associated with Carcinoma in situ, invasion of MEN-2B and NF1. The colon is the second most common lamina propria/muscularis site in the tubular GI tract (after esophagus) for granular mucosae pT1 Tumor invades submucosa Tumor invades lamina cell tumors (Figure 11.37B). Schwannoma and Schwann propria or submucosa cell hamartoma occur in the colon but are relatively rare. and size 2 cm or less Perineuriomas (also referred to as benign fibroblastic pT1a — Tumor size less than 1 cm in polyps, Figure 11.37C) are typically single polyps that greatest dimension pT1b — Tumor size 1 to 2 cm in are found incidentally on screening colonoscopy; there is greatest dimension a slight female predominance. They are often associated pT2 Tumor invades muscularis Same or tumor size >2 cm with serrated polyps. These lesions are occasionally con- propria with invasion of lamina fused with GISTs or other types of neural polyps. propria or submucosa pT3 Tumor invades through the Same muscularis propria into pericolorectal tissues HEMATOLYMPHOID NEOPLASMS pT4 — Tumor invades peritoneum OF THE

COLON or other organs pT4a Tumor penetrates the visceral — peritoneum The colon can be involved by any of the hematolym- pT4b Tumor directly invades or is — phoid neoplasms discussed in earlier chapters. Here, as adherent to other organs or elsewhere, B cell lymphomas far outnumber T cell lym- structures phomas, and diffuse large B cell lymphoma (DLBCL, dis- pN0 No regional lymph node Same metastasis cussed in detail in Chapter 8) is the most common primary pN1 Metastasis in regional lymph colonic lymphoma. A few entities, however, can have a nodes fairly characteristic appearance in this location and will pN1a Metastasis in one regional — be discussed here, including a disease manifestation usu- lymph node pN1b Metastasis in two to three — ally associated with mantle cell lymphoma (MCL) that regional lymph nodes can mimic inherited polyposis syndromes, the so-called pN1c Tumor deposit(s) in — “lymphomatous polyposis”; Langerhans cell histiocytosis the subserosa, or (LCH); and systemic mastocytosis (SM), all of which may nonperitonealized pericolic or perirectal tissues without be encountered in colon biopsies. regional lymph node metastasis pN2a Metastasis in four to six — Mantle Cell Lymphoma regional lymph nodes This lymphoma of small B lymphocytes typically has a pN2b Metastasis in seven or more — regional lymph nodes deceptively low-grade appearance, but is actually relent- pM1 Distant metastasis Same less in its progression and more aggressive than many pM1a Metastasis to single organ or — other small B cell lymphomas. It is closely associated with site (eg, liver, lung, ovary, the t(11;14)(q13;q32) translocation, involving the CCND1 nonregional lymph node) pM1b Metastasis to more than — (cyclin-D1) gene, which comes under the influence of the one organ/site or to the promoter for the immunoglobulin heavy chain (IGH) peritoneum gene. MCL is fairly uncommon, accounting for only a few percentage of non-Hodgkin lymphomas, and tends to occur in middle-aged to older individuals (median age 60 years), with men outnumbering women about two to mucosa covering the lipoma may be unremarkable, hyper- one. The GI tract is the most common extranodal site of plastic, ulcerated, or atrophic. When mucosal ulceration occurrence of MCL, and is involved in about one third of is present, reactive changes can include fibrosis, increased patients with the disease. Common presentations include mitoses, and adipocyte hyperchromasia. Primary liposar- hepatosplenomegaly, lymphadenopathy, and peripheral comas of the colorectum occur but are extremely rare. blood involvement by circulating lymphoma cells. 3 0 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 11.35 This gross photomicrograph shows a leiomyoma arising from the muscularis propria in the colon (A). The tumor is composed of intersecting fascicles with monomorphic spindle cells without significant nuclear atypia, mitoses, or necrosis (B). In the colon, MCL is the lymphoma most commonly lumen. The cells are quite monotonous, but there may be associated with the clinical presentation of lympho- prominent vessels and occasional admixed eosinophilic matous polyposis, although essentially any lymphoma (“pink”) histiocytes. can present with this appearance. This manifestation In addition to the nodular pattern of MCL involve- involves nodular aggregates of MCL that protrude from ment described in the previous paragraph and in Figure the mucosa, creating “polyps” that can look essentially 11.38, this type of lymphoma can have a more diffuse identical to those found in inherited polyposis syndromes pattern, or may surround reactive germinal centers in an such as familial adenomatous polyposis. Histologically, easily overlooked pattern analogous to the normal mantle these nodular aggregates are composed of collections of zones found in other sites of mucosa-associated lymphoid small- or medium-sized lymphocytes with scant cyto- tissue (MALT) such as Peyer’s patches. Mercifully, this plasm and dark nuclei that have irregular, often angu- last pattern is quite rare. In addition to the small cells with lated, contours (Figure 11.38). These collections distort angulated nuclei typically encountered, MCL can also the overlying mucosa, protruding into the intestinal be composed of larger cells that resemble lymphoblasts (A) (B) FIGURE 11.36 Lipomas often arise in the submucosa, as illustrated here in a gross specimen (A). Lipomas are composed of mature adipocytes without atypia (B). 11 Neoplasms of the Colon 3 0 5 (A) (B) (C) FIGURE 11.37 Ganglioneuromas are composed of a mixture of spindled Schwann cells and ganglion cells (A). This granular cell tumor is well-circumscribed (B) and shows polygonal cell borders with granular eosinophilic cytoplasm. Perineuromas are composed of bland spindle cells restricted to the colonic lamina propria (C, courtesy Dr. Rhonda Yantiss; inset, high power) and are often associated with serrated crypts in hyperplastic polyps or SSA/Ps. (the “blastoid” variant) or the cells of DLBCL (the “pleo- be CD5-negative, which can lead to diagnostic confusion. morphic” variant). These variants are associated with a Thankfully, almost all cases of MCL express cyclin-D1 in higher proliferative rate and are considered to be clini- a nuclear pattern and, if the suspicion for MCL is high (eg, cally important, because they are more aggressive than in a patient with atypical lymphoid aggregates in colon typical MCL. biopsies who carries the diagnosis already), this may be Immunophenotypically, MCL consists of CD20- the only stain needed to make the diagnosis. positive B cells that aberrantly coexpress CD5 and, usu- The prognosis for MCL is, unfortunately, relatively ally, CD43. This pattern is similar to that seen in chronic poor, particularly in cases primarily involving the GI lymphocytic leukemia/small lymphocytic lymphoma tract. Median survival is in the range of 3 to 5 years. It is (CLL/SLL). One aid to distinction between the two enti- important to make the distinction between the aggressive ties is CD23, which is often positive in CLL/SLL and nega- variants (blastoid and pleomorphic) and the other hema- tive in MCL. FMC7, which is usually assessed by flow tolymphoid conditions that they mimic, because DLBCL cytometry, has the opposite pattern, being most often pos- and lymphoblastic leukemia/lymphoma are often curable itive in MCL and negative in CLL/SLL. Rarely, MCL can with aggressive therapy, while MCL typically is not. 3 0 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 11.38 Mantle cell lymphoma (MCL) often makes nodular or polypoid collections of monotonous lymphocytes that protrude into the lumen of the GI tract (A). In this case of colonic MCL, there is an erosion/ulcer of the overlying mucosa (arrow). At high magnification (A, inset), the malignant cells exhibit an “angulated” contour and there are admixed, epithelioid histiocytes with eosinophilic cytoplasm (“pink histiocytes,” arrow). Endoscopically (B), the mucosa often has a nodular or polypoid appearance, and the process may mimic inherited polyposis syndromes (so-called “lymphomatous polyposis”). Some cases (C) have a more pleomorphic cytologic appearance. In such cases, MCL may be confused with diffuse large B cell lymphoma or lymphoblastic lymphoma. A “pink histiocyte” is visible at the right of the panel. MCL is immunohistochemically positive for CD5 (D) and nuclear cyclin-D1 (E). Systemic Mastocytosis gene, the D816V mutation being the most common. The WHO classification also sets forth diagnostic criteria for GI involvement by SM is rare, and occurs as part of more the diagnosis of SM (Table 11.8). widespread systemic involvement. Symptoms related to GI GI involvement by SM can be suspected based on involvement are usually nonspecific, including diarrhea, endoscopic findings, particularly in a patient already abdominal pain, and dyspepsia, and are thought to be sec- known to have the disease who develops GI symptoms. ondary to mediator release by the mast cells. According to The mucosa may be nodular, pigmented, or thickened the most recent WHO classification of hematolymphoid and edematous-appearing. Histologically, SM is charac- tumors, SM is a clonal myeloproliferative disorder, and terized by increased numbers of atypical-appearing mast is very commonly associated with mutation of the KIT TABLE 11.8 Diagnostic Criteria for Systemic Mastocytosis Major Criterion Minor Criteria Systemic mastocytosis can be Multifocal, dense infiltrates >25% of mast cells in infiltrates in bone marrow or other extracutaneous diagnosed with the major and of mast cells (≥15 in organ are spindled or have atypical morphology or, of all mast cells in one minor criterion or with at aggregates) in sections of bone marrow aspirate smears, >25% are immature or atypical least three minor criteria bone marrow and/or other Activating point mutation at codon 816 of KIT detected in bone marrow, organs blood, or other extracutaneous organs Expression of CD2 and/or CD25 (in addition to normal mast cell markers) by mast cells in bone marrow, blood, or other extracutaneous organs Serum total tryptase persistently >20 ng/mL (not valid if there is another associated clonal myeloid disorder) Source: Adapted From WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008. 11 Neoplasms of the Colon 3 07 cells, which very often occur in clusters (Figure 11.39). proportion of cases have been found to harbor the BRAF These cells have ovoid or elongated nuclei and can have a V600E mutation. This has opened the door to targeted spindled appearance that can make them difficult to rec- therapy with the BRAF inhibitor vermurafenib. LCH has ognize for what they are. In addition, neoplastic mast cells a variety of other names, including histiocytosis X and often lack the cytoplasmic granules characteristic of their eosinophilic granuloma, and it has several forms that normal counterparts and, when combined with extensive include solitary involvement (usually of bone, lymph node, spindled morphology can make them appear like fibro- or skin), multifocal involvement of one organ system (usu- blasts such as may be seen at the site of healing mucosal ally bones), and multifocal involvement of multiple organ injury. Immunohistochemically, the neoplastic cells of SM systems (usually skin, bone, and visceral organs). The mul- express the typical mast cell antigens KIT and mast cell tifocal, unisystem form has the eponym “Hand–Schüller– tryptase, but can aberrantly express CD2 and/or CD25. Christian disease” and the multifocal, multiorgan form They may also express CD68, which can lead to their con- “Letterer–Siwe disease.” fusion with histiocytes. Histologically, LCH consists of clusters of Langerhans- Mastocytosis has several forms, and the prognosis is like cells, which have ovoid nuclei that often contain a heavily dependent on which type the patient has. Patients longitudinal groove, giving a so-called “coffee bean” with aggressive disease can have a poor prognosis, surviv- appearance. These cells are immunohistochemically posi- ing only a few months. While there is no cure, systemic tive for S100 and CD1a (Figure 11.40) and are usually therapy can be employed for patients with widespread and admixed with numerous inflammatory cells, among which aggressive forms. eosinophils can be very prominent. Ultrastructurally, the cells contain characteristic inclusions (“Birbeck granules”) Langerhans Cell Histiocytosis that have a zipper-like appearance, and which may be dilated at one end like a tennis racket. This rare entity may occasionally make an appearance in The primary importance in recognizing LCH in a GI the GI tract, and can be seen in colon biopsies. Its etiology biopsy is the ability to point the treating physician in the has been controversial, but LCH is now thought to be a direction of the diagnosis so that additional studies may clonal disorder (based on molecular evidence), and a high be performed to look for more widespread involvement. FIGURE 11.39 Systemic mastocytosis (SM) can be difficult to recognize in the GI tract. This case (A) was initially thought to be some type of inflammatory bowel disease before the identification of clusters of unusual-appearing cells (arrow). At high magnification (B), these cells had, in many cases, a spindled appearance (arrow) and there were numerous admixed eosinophils. The cells were immunohistochemically positive for KIT and they had aberrant expression of CD25 (C), establishing the diagnosis of SM. 3 0 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 11.40 Langerhans cell histiocytosis (LCH) is a clonal proliferation of cells with the phenotype of Langerhans cells. They typically have ovoid nuclei (A), many of which contain a longitudinal groove (arrows), imparting a coffee-bean- like appearance. The cells of LCH are positive for S100 protein as well as CD1a (inset). While the diagnosis is most often made without the benefit of electron microscopy, ultrastructural studies reveal characteristic cytoplasmic Birbeck granules (B), which have a zipper-like morphology (electron micrograph, courtesy of Bertram Schnitzer, MD). The prognosis of LCH depends on the extent of involve- ment, with solitary forms having the best outcome and multifocal, multisystem forms the worst, often requiring systemic therapy. The advent of molecular therapy for cases harboring

the BRAF mutation seems to promise new hope for treatment. Rectal Lymphoid Polyps Occasionally in the rectum (and, rarely, in the more proxi- mal colon), biopsies will sample a polypoid mass of benign lymphoid tissue with prominent lymphoid follicles that usu- ally have well-developed germinal centers (Figure 11.41). These benign lymphoid polyps have been branded with the somewhat evocative name of “rectal tonsils” in the lit- erature, and they can mimic malignant lymphoma, par- ticularly MALT lymphoma (discussed in Chapter 8), which FIGURE 11.41 Rectal lymphoid polyps can have an can have reactive germinal centers surrounded by lym- impressive endoscopic appearance, and can raise the phoma cells with a marginal zone phenotype and follicular differential diagnosis of lymphoid neoplasia under the lymphoma (discussed in Chapter 9), where the follicular microscope. At low magnification (A), a cluster of follicular structures themselves are neoplastic. In addition to their structures is seen underlying the distal rectal mucosa. ability to mimic lymphoma, there are scattered reports in While the normal mucosal elements are pushed aside, the literature raising the question of whether these lym- there is no destructive infiltration of the mucosa. At high phoid proliferations may be associated with infectious eti- magnification (B), the follicular structures have reassuring ologies such as Epstein–Barr virus or chlamydia. histologic features, including polarization, with a “dark While the morphology can be striking and these polyps zone” containing tingible-body macrophages (arrow) oriented away from the lumen, and a well-formed mantle can be as large as a centimeter or more, the differential zone (arrowhead) oriented toward the lumen. These benign diagnosis with lymphoma is usually straightforward once lymphoid follicles would be negative for BCL-2 expression. a diagnosis of “rectal tonsil” is entertained. Unlike true Photomicrographs, courtesy of Henry Appelman, MD. 11 Neoplasms of the Colon 3 0 9 MALT lymphomas, there is no destructive infiltration of and may be an important diagnostic consideration the epithelial structures of the mucosa. Well-formed, polar- (Figure 11.42A–D). Tumors from the lung, prostate, ized germinal centers with tingible-body macrophages are ovary, uterus, breast, and pancreas can involve the colon, common, and these structures are negative for BCL-2 on among other primary sites, as well as melanomas. immunohistochemistry, ruling out follicular lymphoma. Müllerian endometrioid adenocarcinomas (Figure 11.42A–B) can be especially difficult to distinguish from well-differentiated CRC. Squamous differentiation is a METASTATIC TUMORS TO THE COLON common feature in Müllerian endometrioid adenocarcino- mas but is quite rare in CRC. Furthermore, endometrioid Although primary colon cancer is common, secondary adenocarcinomas tend to have less cytologic atypia and less involvement of the colon by other tumors does occur “dirty” necrosis compared to CRC. Immunohistochemical (A) (B) (C) (D) FIGURE 11.42 This metastatic endometrioid adenocarcinoma to the colon shows crowded and branching glands (A–B) that mimic colorectal adenocarcinoma. It was positive for CK7, PAX8, ER, and PR, and negative for CK20 and CDX2, supporting the diagnosis. This colon biopsy contains an infiltrating poorly differentiated carcinoma with signet ring cells that fills the lamina propria (C); there is no associated adenomatous component. GATA3 immunostain is positive (D), whereas CK20 and CDX2 are negative, consistent with the patient’s history of previous breast carcinoma. 310 Neoplastic Gastrointestinal Pathology: An Illustrated Guide stains useful in the distinction include CK20 and CDX2 Huang CC, Frankel WL, Doukides T, et al. Prolapse-related changes for colon and CK7, CD10, and PAX8 for Müllerian origin. are a confounding factor in misdiagnosis of sessile serrated adeno- mas in the rectum. Hum Pathol. 2013;44:480–486. Often, there is a known history of an extracolonic Kelly JK. Polypoid prolapsing mucosal folds in diverticular disease. Am tumor and the case is relatively straightforward, but there J Surg Pathol. 1991;15:871–878. are several morphologic features that may help suggest Nakamura S, Kino I, Akagi T. Inflammatory myoglandular polyps of a metastatic tumor rather than a primary CRC. Tumors the colon and rectum. A clinicopathological study of 32 peduncu- lated polyps, distinct from other types of polyps. Am J Surg Pathol. that lack an adenomatous intramucosal component 1992;16:772–779. should raise the diagnostic consideration of a metastasis; Ng KH, Mathur P, Kumarasinghe MP, et al. Cap polyposis: further however, metastatic tumors often overgrow the overly- experience and review. Dis Colon Rectum. 2004;47:1208–1215. ing mucosa and can mimic low- or high-grade dysplasia. Parfitt JR, Shepherd NA. Polypoid mucosal prolapse complicating low rectal adenomas: beware the inflammatory cloacogenic polyp! Growth based deep in the wall rather than the mucosa, Histopathology. 2008;53:91–96. diffuse lymphovascular invasion, pigment (for melanoma), Singh B, Mortensen NJ, Warren BF. Histopathological mimicry in and histology atypical for CRC are all clues indicating mucosal prolapse. Histopathology. 2007;50:97–102. metastases from other primary sites. Immunostains can be extremely useful in the dis- tinction of primary CRC from metastases. The most Conventional Adenoma common immunophenotype for CRC is CK7-/CK20+/ Appelman HD. Con: High-grade dysplasia and villous features should CDX2+. However, several caveats exist in the interpre- not be part of the routine diagnosis of colorectal adenomas. Am J tation of these markers. A significant portion (25%) of Gastroenterol. 2008;103:1329–1331. Bansal M, Fenoglio CM, Robboy SJ, King DW. Are metapla- rectal cancers expresses CK7. Furthermore, up to 13% of sias in colorectal adenomas truly metaplasias? Am J Pathol. rectal tumors are negative for all three markers. A more 1984;115:253–265. recently described antibody that is useful for marking the Brown LJ, Smeeton NC, Dixon MF. Assessment of dysplasia in colorec- intestine is SATB2, which stains approximately 85% of tal adenomas: an observer variation and morphometric study. J Clin Pathol. 1985;38:174–179. CRC. SATB2 is highly specific, with only a small percent- Jensen P, Krogsgaard MR, Christiansen J, et al. Observer variability in age (3%–5%) of breast, ovarian, and lung tumors stain- the assessment of type and dysplasia of colorectal adenomas, ana- ing positive. Additional immunostains that may be useful lyzed using kappa statistics. Dis Colon Rectum. 1995;38:195–198. to confirm tumors of extracolonic origin include TTF1 Lasisi F, Mouchli A, Riddell R, et al. Agreement in interpreting villous elements and dysplasia in adenomas less than one centimetre in (lung), PSA/PSAP/androgen receptor (prostate), PAX8 size. Dig Liver Dis. 2013;45:1049–1055. (ovary, uterus), GATA3/BRST2/mammaglobulin (breast), Levine JS, Ahnen DJ. Clinical practice. Adenomatous polyps of the and SOX10/S100/HMB-45/melan-A (melanoma). As colon. N Engl J Med. 2006;355:2551–2557. always, immunohistochemical stains are best used in a Lieberman DA, Prindiville S, Weiss DG, et al. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic indi- panel and in conjunction with the history and morpho- viduals. JAMA. 2003;290:2959–2967. logic findings. Lieberman DA, Weiss DG, Bond JH, et al. Use of colonoscopy to screen asymptomatic adults for colorectal cancer. Veterans Affairs Cooperative Study Group 380. N Engl J Med. 2000;343:162–168. Lin J, Goldblum JR, Bennett AE, et al. Composite intestinal adenoma- SELECTED REFERENCES microcarcinoid. Am J Surg Pathol. 2012;36:292–295. Mahajan D, Downs-Kelly E, Liu X, et al. Reproducibility of the villous component and high-grade dysplasia in colorectal adenomas <1 Selected references for Hamartomatous Polyps are included cm: implications for endoscopic surveillance. Am J Surg Pathol. in Chapter 6. Selected references for Neuroendocrine 2013;37:427–433. Neoplasms are included in Chapter 3. Additional references Odze RD. Pathology of dysplasia and cancer in inflammatory bowel for Mesenchymal Neoplasms are included in Chapter 5. disease. Gastroenterol Clin North Am. 2006;35:533–552. Pai RK, Rybicki LA, Goldblum JR, et al. Paneth cells in colonic adeno- mas: association with male sex and adenoma burden. Am J Surg Infl ammatory Lesions Pathol. 2013;37:98–103. Rex DK, Goldblum JR. Pro: Villous elements and high-grade dyspla- Chetty R, Bhathal PS, Slavin JL. Prolapse-induced inflammatory pol- sia help guide post-polypectomy colonoscopic surveillance. Am J yps of the colorectum and anal transitional zone. Histopathology. Gastroenterol. 2008;103:1327–1329. 1993;23:63–67. Salaria SN, Abu Alfa AK, Alsaigh NY, et al. Composite intesti- Chiang JM, Changchien CR, Chen JR. Solitary rectal ulcer syndrome: nal adenoma-microcarcinoid clues to diagnosing an under- an endoscopic and histological presentation and literature review. recognised mimic of invasive adenocarcinoma. J Clin Pathol. Int J Colorectal Dis. 2006;21:348–356. 2013;66:302–306. De Petris G, Leung ST. Pseudoneoplasms of the gastrointestinal tract. Terry MB, Neugut AI, Bostick RM, et al. Reliability in the classification Arch Pathol Lab Med. 2010;134:378–392. of advanced colorectal adenomas. Cancer Epidemiol Biomarkers du Boulay CE, Fairbrother J, Isaacson PG. Mucosal prolapse syn- Prev. 2002;11:660–663. drome—a unifying concept for solitary ulcer syndrome and related Torres C, Antonioli D, Odze RD. Polypoid dysplasia and adenomas disorders. J Clin Pathol. 1983;36:1264–1268. in inflammatory bowel disease: a clinical, pathologic, and fol- Guest CB, Reznick RK. Colitis cystica profunda. Review of the litera- low-up study of 89 polyps from 59 patients. Am J Surg Pathol. ture. Dis Colon Rectum. 1989;32:983–988. 1998;22:275–284. 11 Neoplasms of the Colon 311 Ullman T, Odze R, Farraye FA. Diagnosis and management of dysplasia Patil DT, Shadrach BL, Rybicki LA, et al. Proximal colon cancers and in patients with ulcerative colitis and Crohn’s disease of the colon. the serrated pathway: a systematic analysis of precursor histology Inflamm Bowel Dis. 2009;15:630–638. and BRAF mutation status. Mod Pathol. 2012;25:1423–1431. Winawer SJ, Zauber AG, Fletcher RH, et al. Guidelines for colonos- Rex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of the colorec- copy surveillance after polypectomy: a consensus update by the US tum: review and recommendations from an expert panel. Am J Multi-Society Task Force on Colorectal Cancer and the American Gastroenterol. 2012;107:1315–1329. Cancer Society. Gastroenterology. 2006;130:1872–1885. Rosty C, Buchanan DD, Walsh MD, et al. Phenotype and polyp land- scape in serrated polyposis syndrome: a series of 100 patients from Serrated Polyps genetics clinics. Am J Surg Pathol. 2012;36:876–882. Rosty C, Hewett DG, Brown IS, et al. Serrated polyps of the large intes- Aust DE, Baretton GB, Members of the Working Group GI-Pathology tine: current understanding of diagnosis, pathogenesis, and clinical of the German Society of Pathology. Serrated polyps of the colon management. J Gastroenterol. 2013;48:287–302. and rectum (hyperplastic polyps, sessile serrated adenomas, tradi- Salaria SN, Streppel MM, Lee LA, et al. Sessile serrated adenomas: tional serrated adenomas, and mixed polyps)-proposal for diag- high-risk lesions? Hum Pathol. 2012;43:1808–1814. nostic criteria. Virchows Arch. 2010;457:291–297. Schreiner MA, Weiss DG, Lieberman DA. Proximal and large hyper- Bettington M, Walker N, Clouston A, et al. The serrated path- plastic and nondysplastic serrated polyps detected by colo- way to colorectal carcinoma: current concepts and challenges. noscopy are associated with neoplasia. Gastroenterology. Histopathology. 2013;62:367–386. 2010;139:1497–1502. Crowder CD, Sweet K, Lehman A, Frankel WL. Serrated polypo- Sheridan TB, Fenton H, Lewin MR, et al. Sessile serrated adenomas sis is an underdiagnosed and unclear syndrome: the surgical with low- and high-grade dysplasia and early carcinomas: an pathologist has a role in improving detection. Am J Surg Pathol. immunohistochemical study of serrated lesions “caught in the act.” 2012;36:1178–1185. Am J Clin Pathol. 2006;126:564–571. Dhir M, Yachida S, Van Neste L, et al. Sessile serrated adenomas Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the and classical adenomas: an epigenetic perspective on premalig- large intestine: a morphologic and molecular review of an evolving nant neoplastic lesions of the gastrointestinal tract. Int J Cancer. concept. Am J Clin Pathol. 2005;124:380–391. 2011;129:1889–1898. Snover DC. Update on the serrated pathway to colorectal carcinoma. Farris AB, Misdraji J, Srivastava A, et al. Sessile serrated adenoma: Hum Pathol. 2011;42:1–10. challenging discrimination from other serrated colonic polyps. Am Torlakovic E, Skovlund E, Snover DC, et al. Morphologic reappraisal J Surg Pathol. 2008;32:30–35. of serrated colorectal polyps. Am J Surg Pathol. 2003;27:65–81. Goldstein NS. Small colonic microsatellite unstable adenocarcinomas Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. and high-grade epithelial dysplasias in sessile serrated adenoma Gastroenterology. 1996;110:748–755. polypectomy specimens: a study of eight cases. Am J Clin Pathol. Torlakovic EE, Gomez JD, Driman DK, et al. Sessile serrated adenoma 2006;125:132–145. (SSA) vs. traditional serrated adenoma (TSA). Am J Surg Pathol. Iino H, Jass JR, Simms LA, et al. DNA microsatellite instability in 2008;32:21–29. hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol. 1999;52:5–9. Kalady MF, Jarrar A, Leach B, et al. Defining phenotypes and can- Colorectal Adenocarcinoma cer risk in hyperplastic polyposis syndrome. Dis Colon Rectum. 2011;54:164–170. Abdul-Jalil KI, Sheehan KM, Kehoe J, et al. The prognostic value of Kim YH, Kakar S, Cun L, et al. Distinct CpG island methylation tumour regression grade following neoadjuvant chemoradiation profiles and BRAF mutation status in serrated and adenomatous therapy for rectal cancer. Colorectal Dis. 2014;16:O16–O25. colorectal polyps. Int J Cancer. 2008;123:2587–2593. Allam

MF, Lucena RA. Aetiology of sex differences in colorectal can- Leggett B, Whitehall V. Role of the serrated pathway in colorectal can- cer. Eur J Cancer Prev. 2001;10:299–300. cer pathogenesis. Gastroenterology. 2010;138:2088–2100. Arai T, Esaki Y, Sawabe M, et al. Hypermethylation of the hMLH1 pro- Leonard DF, Dozois EJ, Smyrk TC, et al. Endoscopic and surgical man- moter with absent hMLH1 expression in medullary-type poorly agement of serrated colonic polyps. Br J Surg. 2011;98:1685–1694. differentiated colorectal adenocarcinoma in the elderly. Mod Li D, Jin C, McCulloch C, et al. Association of large serrated polyps with Pathol. 2004;17:172–179. synchronous advanced colorectal neoplasia. Am J Gastroenterol. Benson AB, Schrag D, Somerfield MR, et al. American Society of 2009;104:695–702. Clinical Oncology recommendations on adjuvant chemotherapy Lieberman DA, Rex DK, Winawer SJ, et al. Guidelines for colonos- for stage II colon cancer. J Clin Oncol. 2004;22:3408–3419. copy surveillance after screening and polypectomy: a consensus Bond JH. Polyp guideline: diagnosis, treatment, and surveillance for update by the US Multi-Society Task Force on Colorectal Cancer. patients with colorectal polyps. Practice Parameters Committee of Gastroenterology. 2012;143:844–857. the American College of Gastroenterology. Am J Gastroenterol. Limketkai BN, Lam-Himlin D, Arnold MA, Arnold CA. The cut- 2000;95:3053–3063. ting edge of serrated polyps: a practical guide to approach- Bruce WR, Giacca A, Medline A. Possible mechanisms relating diet ing and managing serrated colon polyps. Gastrointest Endosc. and risk of colon cancer. Cancer Epidemiol Biomarkers Prev. 2013;77:360–375. 2000;9:1271–1279. Lu FI, van Niekerk dW, Owen D, et al. Longitudinal outcome study Burt RW. Colon cancer screening. Gastroenterology. 2000;119:837–853. of sessile serrated adenomas of the colorectum: an increased risk Chang GJ, Rodriguez-Bigas MA, Skibber JM, Moyer VA. Lymph node for subsequent right-sided colorectal carcinoma. Am J Surg Pathol. evaluation and survival after curative resection of colon cancer: 2010;34:927–934. systematic review. J Natl Cancer Inst. 2007;99:433–441. Pai RK, Hart J, Noffsinger AE. Sessile serrated adenomas strongly pre- Chetty R, Gill P, Govender D, et al. International study group on rectal dispose to synchronous serrated polyps in non-syndromic patients. cancer regression grading: interobserver variability with commonly Histopathology. 2010;56:581–588. used regression grading systems. Hum Pathol. 2012;43:1917–1923. Pai RK, Mojtahed A, Rouse RV, et al. Histologic and molecular anal- Compton C, Fenoglio-Preiser CM, Pettigrew N, Fielding LP. yses of colonic perineurial-like proliferations in serrated polyps: American Joint Committee on Cancer Prognostic Factors perineurial-like stromal proliferations are seen in sessile serrated Consensus Conference: Colorectal Working Group. Cancer. adenomas. Am J Surg Pathol. 2011;35:1373–1380. 2000;88:1739–1757. 312 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Connelly JH, Robey-Cafferty SS, Cleary KR. Mucinous carcinomas of Jessurun J, Romero-Guadarrama M, Manivel JC. Medullary adeno- the colon and rectum. An analysis of 62 stage B and C lesions. Arch carcinoma of the colon: clinicopathologic study of 11 cases. Hum Pathol Lab Med. 1991;115:1022–1025. Pathol. 1999;30:843–848. Cooper HS, Deppisch LM, Gourley WK, et al. Endoscopically removed Jiang W, Roma AA, Lai K, et al. Endometriosis involving the mucosa malignant colorectal polyps: clinicopathologic correlations. of the intestinal tract: a clinicopathologic study of 15 cases. Mod Gastroenterology. 1995;108:1657–1665. Pathol. 2013;26:1270–1278. Cranley JP, Petras RE, Carey WD, et al. When is endoscopic polypec- Jin M, Roth R, Rock JB, et al. The Impact of Tumor Deposits on tomy adequate therapy for colonic polyps containing invasive car- Colonic Adenocarcinoma AJCC TNM Staging and Outcome. Am cinoma? Gastroenterology. 1986;91:419–427. J Surg Pathol. 2014;39(1):109–115. de Campos-Lobato LF, Stocchi L, de Sousa JB, et al. Less than 12 nodes Kakar S, Aksoy S, Burgart LJ, Smyrk TC. Mucinous carcinoma of the in the surgical specimen after total mesorectal excision following colon: correlation of loss of mismatch repair enzymes with clinico- neoadjuvant chemoradiation: it means more than you think! Ann pathologic features and survival. Mod Pathol. 2004;17:696–700. Surg Oncol. 2013;20:3398–3406. Keshava A, Chapuis PH, Chan C, et al. The significance of involve- De Lott LB, Morrison C, Suster S, et al. CDX2 is a useful marker ment of a free serosal surface for recurrence and survival follow- of intestinal-type differentiation: a tissue microarray-based ing resection of clinicopathological stage B and C rectal cancer. study of 629 tumors from various sites. Arch Pathol Lab Med. Colorectal Dis. 2007;9:609–618. 2005;129:1100–1105. Kojima M, Nakajima K, Ishii G, et al. Peritoneal elastic laminal inva- Dillman RO, Aaron K, Heinemann FS, McClure SE. Identification of sion of colorectal cancer: the diagnostic utility and clinicopatho- 12 or more lymph nodes in resected colon cancer specimens as an logic relationship. Am J Surg Pathol. 2010;34:1351–1360. indicator of quality performance. Cancer. 2009;115:1840–1848. Kojima M, Shimazaki H, Iwaya K, et al. Practical utility and objec- Edge S, Byrd DR, Compton cc, et al. Colon and Rectum. AJCC Cancer tivity: does evaluation of peritoneal elastic laminal invasion in Staging Manual. 7th ed:New York, NY; Springer-Verlag; 2010. colorectal cancer overcome these contrary problems? Am J Surg Fenoglio CM, Kaye GI, Lane N. Distribution of human colonic lymphat- Pathol. 2014;38:144–145. ics in normal, hyperplastic, and adenomatous tissue. Its relationship Kojima M, Yokota M, Saito N, et al. Elastic laminal invasion in colon to metastasis from small carcinomas in pedunculated adenomas, cancer: diagnostic utility and histological features. Front Oncol. with two case reports. Gastroenterology. 1973;64:51–66. 2012;2:179. Garfinkel L, Mushinski M. U.S. cancer incidence, mortality and sur- Le Voyer TE, Sigurdson ER, Hanlon AL, et al. Colon cancer survival vival: 1973–1996. Stat Bull Metrop Insur Co. 1999;80:23–32. is associated with increasing number of lymph nodes analyzed: Gleisner AL, Mogal H, Dodson R, et al. Nodal status, number of a secondary survey of intergroup trial INT-0089. J Clin Oncol. lymph nodes examined, and lymph node ratio: what defines prog- 2003;21:2912–2919. nosis after resection of colon adenocarcinoma? J Am Coll Surg. Liang WY, Chang WC, Hsu CY, et al. Retrospective evaluation of elas- 2013;217:1090–1100. tic stain in the assessment of serosal invasion of pT3N0 colorectal Goldstein NS, Long A, Kuan SF, Hart J. Colon signet ring cell ade- cancers. Am J Surg Pathol. 2013;37:1565–1570. nocarcinoma: immunohistochemical characterization and com- Lipper S, Kahn LB, Ackerman LV. The significance of microscopic inva- parison with gastric and typical colon adenocarcinomas. Appl sive cancer in endoscopically removed polyps of the large bowel. A Immunohistochem Mol Morphol. 2000;8:183–188. clinicopathologic study of 51 cases. Cancer. 1983;52:1691–1699. Goldstein NS, Turner JR. Pericolonic tumor deposits in patients with Ludeman L, Shepherd NA. Serosal involvement in gastrointesti- T3N+MO colon adenocarcinomas: markers of reduced disease free nal cancer: its assessment and significance. Histopathology. survival and intra-abdominal metastases and their implications for 2005;47:123–131. TNM classification. Cancer. 2000;88:2228–2238. McCluggage WG, Clements WD. Endosalpingiosis of the colon and Goldstein NS. Lymph node recoveries from 2427 pT3 colorectal appendix. Histopathology. 2001;39:645–646. resection specimens spanning 45 years: recommendations for a Medani M, Kelly N, Samaha G, et al. An appraisal of lymph node ratio minimum number of recovered lymph nodes based on predictive in colon and rectal cancer: not one size fits all. Int J Colorectal Dis. probabilities. Am J Surg Pathol. 2002;26:179–189. 2013;28:1377–1384. Gopal P, Lu P, Ayers GD, et al. Tumor deposits in rectal adenocarci- Miller ED, Robb BW, Cummings OW, Johnstone PA. The effects of noma after neoadjuvant chemoradiation are associated with poor preoperative chemoradiotherapy on lymph node sampling in rectal prognosis. Mod Pathol. 2014;27:1281–1287. cancer. Dis Colon Rectum. 2012;55:1002–1007. Govindarajan A, Gönen M, Weiser MR, et al. Challenging the feasibil- Min BS, Kim NK, Ko YT, et al. Clinicopathological features of signet- ity and clinical significance of current guidelines on lymph node ring cell carcinoma of the colon and rectum: a case-matched study. examination in rectal cancer in the era of neoadjuvant therapy. Hepatogastroenterology. 2009;56:984–988. J Clin Oncol. 2011;29:4568–4573. Moon SH, Kim DY, Park JW, et al. Can the new American Joint Greene FL. Epithelial misplacement in adenomatous polyps of the colon Committee on Cancer staging system predict survival in rectal can- and rectum. Cancer. 1974;33:206–217. cer patients treated with curative surgery following preoperative Grin A, Messenger DE, Cook M, et al. Peritoneal elastic lamina inva- chemoradiotherapy? Cancer. 2012;118:4961–4968. sion: limitations in its use as a prognostic marker in stage II Morson BC, Whiteway JE, Jones EA, et al. Histopathology and prog- colorectal cancer. Hum Pathol. 2013;44:2696–2705. nosis of malignant colorectal polyps treated by endoscopic polyp- Gunderson LL, Jessup JM, Sargent DJ, et al. Revised tumor and node ectomy. Gut. 1984;25:437–444. categorization for rectal cancer based on surveillance, epidemiol- Moug SJ, Oliphant R, Balsitis M, et al. The lymph node ratio opti- ogy, and end results and rectal pooled analysis outcomes. J Clin mises staging in patients with node positive colon cancer with Oncol. 2010;28:256–263. implications for adjuvant chemotherapy. Int J Colorectal Dis. Hase K, Shatney C, Johnson D, et al. Prognostic value of tumor “bud- 2014;29:599–604. ding” in patients with colorectal cancer. Dis Colon Rectum. Muto T, Bussey HJ, Morson BC. Pseudo-carcinomatous invasion 1993;36:627–635. in adenomatous polyps of the colon and rectum. J Clin Pathol. Jass JR. Classification of colorectal cancer based on correlation of 1973;26:25–31. clinical, morphological and molecular features. Histopathology. Nagtegaal ID, Quirke P. Colorectal tumour deposits in the mesorectum 2007;50:113–130. and pericolon; a critical review. Histopathology. 2007;51:141–149. Jass JR. Hereditary Non-Polyposis Colorectal Cancer: the rise and Panarelli NC, Schreiner AM, Brandt SM, et al. Histologic features fall of a confusing term. World J Gastroenterol. 2006;12:4943– and cytologic techniques that aid pathologic stage assessment of 4950. colonic adenocarcinoma. Am J Surg Pathol. 2013;37:1252–1258. 11 Neoplasms of the Colon 313 Parfitt JR, Driman DK. The total mesorectal excision specimen for rec- Wang HS, Liang WY, Lin TC, et al. Curative resection of T1 colorectal tal cancer: a review of its pathological assessment. J Clin Pathol. carcinoma: risk of lymph node metastasis and long-term prognosis. 2007;60:849–855. Dis Colon Rectum. 2005;48:1182–1192. Puppa G, Maisonneuve P, Sonzogni A, et al. Pathological assessment of Washington MK, Berlin J, Branton P, et al. Protocol for the examina- pericolonic tumor deposits in advanced colonic carcinoma: relevance tion of specimens from patients with primary carcinoma of the to prognosis and tumor staging. Mod Pathol. 2007;20:843–855. colon and rectum. Arch Pathol Lab Med. 2009;133:1539–1551. Puppa G, Shepherd NA, Sheahan K, Stewart CJ. Peritoneal elastic lam- Washington MK, Berlin J, Branton P, et al. Protocol for the examina- ina invasion in colorectal cancer: the answer to a controversial area tion of specimens from patients with primary carcinoma of the of pathology? Am J Surg Pathol. 2011;35:465–469. colon and rectum. Arch Pathol Lab Med. 2009;133:1539–1551. Puppa G, Sonzogni A, Colombari R, Pelosi G. TNM staging system Wünsch K, Müller J, Jähnig H, et al. Shape is not associated with the origin of colorectal carcinoma: a critical appraisal of challenging issues. of pericolonic tumor deposits. Am J Clin Pathol. 2010;133:388–394. Arch Pathol Lab Med. 2010;134:837–852. Xie J, Itzkowitz SH. Cancer in inflammatory bowel disease. World J Puppa G, Ueno H, Kayahara M, et al. Tumor deposits are encoun- Gastroenterol. 2008;14:378–389. tered in advanced colorectal cancer and other adenocarcinomas: Zhang J, Lv L, Ye Y, et al. Comparison of metastatic lymph node ratio an expanded classification with implications for colorectal cancer staging system with the 7th AJCC system for colorectal cancer. staging system including a unifying concept of in-transit metasta- J Cancer Res Clin Oncol. 2013;139:1947–1953. ses. Mod Pathol. 2009;22:410–415. Puppa G. Enhanced pathologic analysis for pericolonic tumor deposits: is it worth it? Am J Clin Pathol. 2010;134:1019–1021. Mesenchymal Tumors (See Also Chapter 5) Rock JB, Washington MK, Adsay NV, et al. Debating deposits: an Eslami-Varzaneh F, Washington K, Robert ME, et al. Benign fibroblas- interobserver variability study of lymph nodes and pericolonic tic polyps of the colon: a histologic, immunohistochemical, and tumor deposits in colonic adenocarcinoma. Arch Pathol Lab Med. ultrastructural study. Am J Surg Pathol. 2004;28:374–378. 2014;138:636–642. Gibson JA, Hornick JL. Mucosal Schwann cell “hamartoma”: clinico- Rüschoff J, Dietmaier W, Lüttges J, et al. Poorly differentiated colonic pathologic study of 26 neural colorectal polyps distinct from neurofi- adenocarcinoma, medullary type: clinical, phenotypic, and molec- bromas and mucosal neuromas. Am J Surg Pathol. 2009;33:781–787. ular characteristics. Am J Pathol. 1997;150:1815–1825. Groisman GM, Polak-Charcon S. Fibroblastic polyp of the colon and Ryan R, Gibbons D, Hyland JM, et al. Pathological response following colonic perineurioma: 2 names for a single entity? Am J Surg long-course neoadjuvant chemoradiotherapy for locally advanced Pathol. 2008;32:1088–1094. rectal cancer. Histopathology. 2005;47:141–146. Hancock BJ, Vajcner A. Lipomas of the colon: a clinicopathologic Seitz U, Bohnacker S, Seewald S, et al. Is endoscopic polypectomy an review. Can J Surg. 1988;31:178–181. adequate therapy for malignant colorectal adenomas? Presentation

Hornick JL, Fletcher CD. Soft tissue perineurioma: clinicopathologic of 114 patients and review of the literature. Dis Colon Rectum. analysis of 81 cases including those with atypical histologic fea- 2004;47:1789–1797. tures. Am J Surg Pathol. 2005;29:845–858. Shepherd NA, Baxter KJ, Love SB. Influence of local peritoneal involve- Mendelsohn G, Diamond MP. Familial ganglioneuromatous polyposis ment on pelvic recurrence and prognosis in rectal cancer. J Clin of the large bowel. Report of a family with associated juvenile pol- Pathol. 1995;48:849–855. yposis. Am J Surg Pathol. 1984;8:515–520. Shepherd NA, Baxter KJ, Love SB. The prognostic importance of peri- Miettinen M, Furlong M, Sarlomo-Rikala M, et al. Gastrointestinal toneal involvement in colonic cancer: a prospective evaluation. stromal tumors, intramural leiomyomas, and leiomyosarcomas Gastroenterology. 1997;112:1096–1102. in the rectum and anus: a clinicopathologic, immunohistochemi- Shia J, Klimstra DS, Bagci P, et al. TNM staging of colorectal carci- cal, and molecular genetic study of 144 cases. Am J Surg Pathol. noma: issues and caveats. Semin Diagn Pathol. 2012;29:142–153. 2001;25:1121–1133. Shinto E, Ueno H, Hashiguchi Y, et al. The subserosal elastic lamina: Miettinen M, Sarlomo-Rikala M, Sobin LH. Mesenchymal tumors of an anatomic landmark for stratifying pT3 colorectal cancer. Dis muscularis mucosae of colon and rectum are benign leiomyomas Colon Rectum. 2004;47:467–473. that should be separated from gastrointestinal stromal tumors—a Snaebjornsson P, Coupe VM, Jonasson L, et al. pT4 stage II and III clinicopathologic and immunohistochemical study of eighty-eight colon cancers carry the worst prognosis in a nationwide survival cases. Mod Pathol. 2001;14:950–956. analysis. Shepherd’s local peritoneal involvement revisited. Int J Miettinen M, Shekitka KM, Sobin LH. Schwannomas in the colon and Cancer. 2014;135:467–478. rectum: a clinicopathologic and immunohistochemical study of 20 Soga K, Konishi H, Tatsumi N, et al. Clear cell adenocarcinoma of the cases. Am J Surg Pathol. 2001;25:846–855. colon: a case report and review of literature. World J Gastroenterol. Rogy MA, Mirza D, Berlakovich G, et al. Submucous large-bowel lipo- 2008;14:1137–1140. mas—presentation and management. An 18-year study. Eur J Surg. Swamy R. Histopathological reporting of pT4 tumour stage in colorec- 1991;157:51–55. tal carcinomas: dotting the ‘i’s and crossing the ‘t’s. J Clin Pathol. Shekitka KM, Sobin LH. Ganglioneuromas of the gastrointestinal 2010;63:110–115. tract. Relation to Von Recklinghausen disease and other multiple Taliano RJ, LeGolvan M, Resnick MB. Immunohistochemistry of tumor syndromes. Am J Surg Pathol. 1994;18:250–257. colorectal carcinoma: current practice and evolving applications. Singhi AD, Montgomery EA. Colorectal granular cell tumor: a clinico- Hum Pathol. 2013;44:151–163. pathologic study of 26 cases. Am J Surg Pathol. 2010;34:1186–1192. Ueno H, Hashiguchi Y, Shimazaki H, et al. Peritumoral deposits as an adverse prognostic indicator of colorectal cancer. Am J Surg. 2014;207:70–77. Hematolymphoid Tumors Ueno H, Mochizuki H, Hashiguchi Y, et al. Risk factors for an adverse outcome in early invasive colorectal carcinoma. Gastroenterology. Behdad A, Owens SR. Langerhans cell histiocytosis involving the gas- 2004;127:385–394. trointestinal tract. Arch Pathol Lab Med. 2014;138:1350–1352. Ueno H, Mochizuki H, Shirouzu K, et al. Multicenter study for optimal Behdad A, Owens SR. Systemic mastocytosis involving the gastro- categorization of extramural tumor deposits for colorectal cancer intestinal tract: case report and review. Arch Pathol Lab Med. staging. Ann Surg. 2012;255:739–746. 2013;137:1220–1223. Volk EE, Goldblum JR, Petras RE, et al. Management and outcome Burke JS. Lymphoproliferative disorders of the gastrointestinal tract: a of patients with invasive carcinoma arising in colorectal polyps. review and pragmatic guide to diagnosis. Arch Pathol Lab Med. Gastroenterology. 1995;109:1801–1807. 2011;135:1284–1297. 314 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Cramer SF, Romansky S, Hulbert B, et al. The rectal tonsil: a reaction lymphoid tissue lymphoma: a report of three cases. Pathol Res Pract. to chlamydial infection? Am J Surg Pathol. 2009;33:483–485. 2005;201:757–761. Detlefsen S, Fagerberg CR, Ousager LB, et al. Histiocytic disorders of O’Malley DP, Goldstein NS, Banks PM. The recognition and classifi- the gastrointestinal tract. Hum Pathol. 2013;44:683–696. cation of lymphoproliferative disorders of the gut. Hum Pathol. Doyle LA, Hornick JL. Pathology of extramedullary mastocytosis. 2014;45:899–916. Immunol Allergy Clin North Am. 2014;34:323–339. Skarbnik AP, Goy AH. Mantle cell lymphoma: state of the art. Clin Adv Doyle LA, Sepehr GJ, Hamilton MJ, et al. A clinicopathologic study Hematol Oncol. 2015;13:44–55. of 24 cases of systemic mastocytosis involving the gastrointesti- nal tract and assessment of mucosal mast cell density in irritable bowel syndrome and asymptomatic patients. Am J Surg Pathol. Metastatic Tumors 2014;38:832–843. Farris AB, Lauwers GY, Ferry JA, Zukerberg LR. The rectal tonsil: a Disibio G, French SW. Metastatic patterns of cancers: results from a reactive lymphoid proliferation that may mimic lymphoma. Am J large autopsy study. Arch Pathol Lab Med. 2008;132:931–939. Surg Pathol. 2008;32:1075–1079. Estrella JS, Wu TT, Rashid A, Abraham SC. Mucosal colonization Foukas PG, de Leval L. Recent advances in intestinal lymphomas. by metastatic carcinoma in the gastrointestinal tract: a potential Histopathology. 2015;66:112–136. mimic of primary neoplasia. Am J Surg Pathol. 2011;35:563–572. Hashimoto Y, Omura H, Tanaka T, et al. CD5-negative mantle cell Magnusson K, de Wit M, Brennan DJ, et al. SATB2 in combination lymphoma resembling extranodal marginal zone lymphoma of with cytokeratin 20 identifies over 95% of all colorectal carcino- mucosa-associated lymphoid tissue: a case report. J Clin Exp mas. Am J Surg Pathol. 2011;35:937–948. Hematop. 2012;52:185–191. Samo S, Sherid M, Husein H, et al. Metastatic malignant melanoma to Hawkes EA, Wotherspoon A, Cunningham D. Diagnosis and man- the colon: a case report and review of the literature. J Gastrointest agement of rare gastrointestinal lymphomas. Leuk Lymphoma. Cancer. 2014;45:221–224. 2012;53:2341–2350. Yang Q, Wang H, Cho HY, et al. Carcinoma of müllerian origin pre- Kojima M, Itoh H, Motegi A, et al. Localized lymphoid hyper- senting as colorectal cancer: a clinicopathologic study of 13 Cases. plasia of the rectum resembling polypoid mucosa-associated Ann Diagn Pathol. 2011;15:12–18. 12 Neoplasms of the Anus SCOT T R . OW ENS INTRODUCTION biopsied and encountered by the diagnostic pathologist. Most common are squamous dysplasias and carcinomas, The anus/anal canal is the site of a handful of diseases which are pathophysiologically related to squamous con- that pathologists encounter relatively infrequently. ditions elsewhere in the anogenital region, and which are Nonetheless, it is an important site of disease in that its frequently the result of infection by human papillomavi- anatomically restricted space leaves little room for mass- rus (HPV). The classification of squamous dysplasia has forming processes to grow before causing relatively sig- evolved to match that in the male and female genitalia, nificant symptoms. In addition, the anal region is subject adopting the “squamous intraepithelial lesion” terminol- to squamous dysplasias and neoplasms that are identical ogy that will be discussed in the Squamous Dysplasia sec- to their counterparts in the genitalia. Finally, the meeting tion. Less common lesions include adenocarcinomas arising of several different tissue types (columnar rectal mucosa, from the anal gland/duct epithelium, which must be distin- anal glands, squamous mucosa, and skin) leads to a num- guished from adenocarcinomas that arise from the distal ber of unique disease entities that can affect this small rectum and involve the anus by distal extension. The anus anatomic location. can be involved by extramammary Paget disease, either pri- The anal canal develops from a mixture of endoderm, mary (similar to the vulva) or associated with underlying comprising the upper two thirds of the canal, and ecto- colorectal neoplasia, and it can be the site of primary mela- derm, comprising the lower third. The division between noma. Finally, tumors of the perianal skin, such as basal these epithelial origins is the dentate line, which is nor- cell carcinoma, can appear as anal lesions, and can present mally the dividing line between rectal mucosa above and a diagnostic challenge in their separation from squamous squamous mucosa below. At the junction between these carcinomas with a “basaloid” histologic appearance. epithelial types, there is usually a hybrid “transitional” mucosa that appears histologically as a mixture of strati- fied squamoid cells and more columnar mucus-producing BENIGN CONDITIONS AND TUMOR MIMICS cells (Figure 12.1). In some specimens from this region, the transitional mucosa can be difficult to find, because it can Several conditions can result in tumor- or mass-like lesions be very focal and/or overgrown by more distal squamous in the anal canal, and are worth discussing in the differen- epithelium secondary to prolapse changes (Figure 12.2). tial diagnosis of anal malignancies. Perhaps the most clin- Anal glands (or ducts) are lined by similar transitional- ically ominous-appearing of these are the inflammatory type epithelium, and secrete into the canal near the den- masses that can result from prolapse of the rectal mucosa tate line (Figure 12.3A–B). just above the anal canal (also known as “inflammatory The complex anatomy and histology of this small cloacogenic polyp;” see also Chapter 11). Other masses region of the gastrointestinal (GI) tract can give rise to sev- and nodules in and around the anus can be caused by hem- eral different types of malignancy, any of which may be orrhoids and fibroepithelial “tags.” 315 316 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 12.1 The anal transition zone contains a FIGURE 12.3 The anal glands have a variable appearance, “hybrid” epithelium, with stratified squamous-like cells and can be found in the subepithelial tissues surrounding that are admixed with mucus-producing cells (arrows). the anal canal in the region of the dentate line. They can This type of epithelium occupies the zone between the have an adnexal-like appearance (A), or appear larger and anal squamous mucosa below and the colonic mucosa more duct-like (B). They are lined by epithelium with similar above, and is variable in length from person to person. characteristics to that of the anal transition zone. Hemorrhoids situations leading to increased intra-abdominal pressure such as pregnancy, ascites, prolonged sitting, and strain- These dilated submucosal vessels, part of the arteriovenous ing at stool. Their incidence is difficult to ascertain, but complex of the anal region, often arise in the same clinical they are thought to affect at least 5% of the population. setting(s) as mucosal prolapse, including conditions and They can affect the mucosa/submucosa proximal (inter- nal hemorrhoids) or distal (external hemorrhoids) to the dentate line. The venous plexuses that give rise to these dilated vessels are referred to as the superior and inferior hemorrhoidal plexuses, reflecting how often this condition occurs. Grossly, hemorrhoids are not usually a diagnostic challenge and are mentioned in many endoscopic reports, typically occurring in the left and right posterolateral and right anterior anus. Histologically, they are characterized by dilated submucosal vessels, often containing thrombi in varying states of organization (Figure 12.4A–B). The overlying squamous epithelium frequently has prolapse- related hyperparakeratosis as well. While hemorrhoids are typically innocuous, other dis- eases that will be discussed in detail later may complicate them, and it is important to keep these things in mind when grossing and when addressing yet another seemingly uninspiring “hemorrhoid” under the microscope. First, anything that appears as a mass in the anal canal, includ- ing carcinomas and melanomas, can mimic a hemorrhoid FIGURE 12.2 In some cases, the transition zone is and may be excised for that clinical indication, appearing difficult or impossible to find, often because of overgrowth unexpectedly when the tissue is examined histologically. of squamous epithelium secondary to prolapse. In this In addition, squamous dysplasias, usually related to HPV, photomicrograph, the colonic mucosa on the right directly can complicate bona fide hemorrhoids (Figure 12.5). This abuts the anal squamous mucosa on the left, without the can range from low-grade condylomatous lesions to carci- characteristic transitional epithelium. There is even some nomas in situ, and is perhaps more easily overlooked than overgrowth of squamous cells superficial to colonic crypts. invasive carcinoma or melanoma because the other typical 12 Neoplasms of the Anus 317 FIGURE 12.4 Hemorrhoids are composed of dilated and blood-filled submucosal vessels that can occur proximal (internal hemorrhoids) or distal (external hemorrhoids) to the dentate line. In (A), a collection of hemorrhoidal vessels underlies the anal squamous mucosa on the right, and the transition zone on the left. Transitional epithelium is seen in the middle of the image (arrow). Hemorrhoids commonly contain evidence of thrombosis and organization (B), where ingrowth of fibroblasts is seen at the periphery of several vessels. features of hemorrhoids are present. Additional discussion of the various entities that can mimic or complicate hem- orrhoids is present in subsequent sections. Anal Tags/Fibroepithelial Polyps These benign lesions consist of polypoid protrusions of anal squamous epithelium and underlying fibrovascular tissue, and are

commonly confused with hemorrhoids on clinical examination. They are also known as “hypertro- phied anal papillae.” These lesions consist of hyalinized subepithelial connective tissue with overlying squamous epithelium (Figure 12.6A–B). They lack the characteristic dilated submucosal vessels of hemorrhoids, and can have reactive-appearing myofibroblasts in their stroma. Anal tags are essentially identical to cutaneous fibroepithelial polyps (also known as acrochordons). SQUAMOUS DYSPLASIA FIGURE 12.5 Other conditions affecting the anal canal Epidemiology and Pathogenesis can complicate or be superimposed on hemorrhoids. In Squamous dysplasia of the anus is relatively uncommon this case, a dilated hemorrhoidal vessel at the bottom of compared to the incidence in the female genital tract, the image is beneath an HPV-related condyloma. but its incidence has risen over recent decades. There is a 318 Neoplastic Gastrointestinal Pathology: An Illustrated Guide The pathogenesis of HPV-related squamous disease involves direct effects of the virus on patients’ genomes, with integration of HPV DNA into the host genetic mate- rial in cases related to high-risk genotypes that ultimately lead to malignancy. Low-risk types may remain uninte- grated in host cells as episomes. The HPV-associated oncoproteins E5, E6, and E7 are thought to be involved in malignant transformation, via inactivation of the tumor suppressor proteins p53 (by E6) and pRb (by E7). E5 may have an effect on growth factor receptors, further pro- moting proliferation. The upregulation of p16 that results from pRb inactivation underlies the use of p16 immuno- histochemistry in the diagnosis of HPV-related squamous dysplasia and neoplasia, with “block-like” p16 positivity considered a surrogate marker for HPV (Figure 12.7). FIGURE 12.6 Anal tags are histologically identical Terminology to fibroepithelial polyps in the skin, with a core of fibrovascular tissue that varies from loose and myxoid (A) The terminology of anogenital squamous lesions has his- to more dense and hyalinized (B). There is often overlying torically been confusing and nonstandardized, ranging squamous epithelium that may have hyperparakeratosis from terms originally employed in the context of cervical associated with mechanical irritation, as in (B). Pap smear cytology and later carried over to biopsy pathol- ogy, to language adapted from squamous skin cancer and applied to anogenital mucosae. In 2012, a project jointly undertaken by the College of American Pathologists slight female predominance overall, but the incidence of (CAP) and the American Society for Colposcopy and anal condylomas, other forms of intraepithelial neoplasia, Cervical Pathology (ASCCP) recommended a standard- and squamous cell carcinoma are much higher in homo- ized terminology for anogenital squamous lesions. This sexual men who practice anal receptive intercourse and/or CAP–ASCCP Lower Anogenital Squamous Terminology are HIV positive. In addition, these lesions can complicate (LAST) project recommends dividing anal squamous other processes found in the anus, such as hemorrhoids or dysplasias into a two-tiered naming/grading convention, anal tags. Its recognition as a sexually transmitted process related to HPV infection has shed new light on its diag- nosis, and has resulted in attempts at screening programs such as anal Pap smears for high-risk populations, includ- ing immunosuppressed patients such as those with HIV, and patients with other sexually transmitted infections. Patients with HPV-related disease in the genital region, including the vulva and cervix, are at high risk for anal neoplasia as well. As with genital HPV-related disease, certain HPV genotypes are considered “high-risk” for inducing the development of anal neoplasms. The most common geno- types infecting this region are 6, 11, 16, and 18, and types 16 and 18 are the most commonly encountered high-risk types. Other high-risk genotypes include 31, 33, and 35. Although these genotypes have the highest likelihood of leading to anogenital neoplasia, the majority of infected patients do not go on to develop squamous carcinoma. Instead, cell-mediated immunity keeps the infection in check for most patients, and a humoral immune response provides permanent immunity. In recent years, this fact FIGURE 12.7 Immunohistochemistry for p16 is useful as has been exploited to develop vaccines against the com- a surrogate marker for HPV-driven squamous dysplasia in mon high-risk genotypes of HPV. “Low-risk” types, such the anal canal. Dense, block-like nuclear and cytoplasmic as 6 and 11, are associated with squamous dysplasia but positivity as in the left half of the image is indicative of have a much lower risk of progression to malignancy. HPV-related dysplasia. 12 Neoplasms of the Anus 319 based on the presence of low-grade or high-grade dyspla- the lower 2/3. P/AIN3 demonstrates full-thickness atypia/ sia. Low-grade squamous intraepithelial neoplasia (LSIL) dysplasia, effectively “carcinoma in situ.” The designation is most often related to low-risk HPV genotypes such as of HSIL (AIN3) subsumes the now-out-of-date term of 6 and 11, while its high-grade counterpart (high-grade “Bowen’s disease.” squamous intraepithelial neoplasia [HSIL]) and invasive squamous carcinoma tend to be associated with the high- Clinical and Macroscopic Features risk types including 16 and 18. The LAST project also provides recommendations on how and when to use p16 Patients typically present with bleeding, pain, and itching. immunohistochemistry in the diagnosis and grading of In addition to the macroscopic appearance of condylo- these lesions. mata, squamous lesions of the anus may present clinically While anal dysplasias are currently divided into as white, pigmented, or erythematous areas. There may low- and high-grade tiers, the older terminology of “anal also be papules or scaly plaques. As in the cervix, acetic intraepithelial neoplasia” (AIN) and “perianal intraepi- acid may be used to highlight the lesions, imparting an thelial neoplasia” (PAIN) linger in the system of nomen- “acetowhite” appearance that may help in clinical iden- clature recommended by the LAST project. The former tification. Biopsies from anal tags, hemorrhoids, or other is used for lesions in squamous mucosa of the anal canal protrusions in the anal canal may come with a request to itself, while the latter refers to lesions affecting the peri- “rule out condyloma,” and these may either mimic or hide anal skin within 5 cm of the anal verge. These older terms true HPV-related dysplasia. reflect a division of squamous dysplasia into three tiers or grades, with LSIL encompassing grade 1 and HSIL grades Microscopic Features 2 and 3. According to the LAST criteria, anal squamous lesions are to be reported using the LSIL/HSIL nomen- The dysplastic cells in these cases are characterized by clature, followed by the P/AIN grade in parenthesis; for nuclear irregularity, hyperchromasia, enlargement, and example, “LSIL (AIN1).” Histologically, P/AIN1 is charac- pleomorphism (Figure 12.8A–D). In addition, particu- terized by squamous atypia and dysmaturation restricted larly in examples of LSIL, there may be histologic evi- to the basal 1/3 of the epithelium, and P/AIN2 to roughly dence of HPV infection in the form of koilocytic changes. FIGURE 12.8 As in other sites invested with squamous epithelium, dysplasia in the anal canal is characterized by disorganized, immature cells that have pleomorphic and hyperchromatic nuclei (A). Mitotic figures (arrow), normally restricted to the basal layer of proliferative cells, are easily identifiable above the basal layer. In high-grade squamous intraepithelial lesions (HSIL), there is full-thickness atypia and a lack of maturation toward the surface of the epithelium (B). HSIL comprises anal intraepithelial neoplasia grades 2 (C) and 3 (D), where immature cells and mitotic activity (arrows) reach the middle third and upper third of the epithelium, respectively. 3 2 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 12.9 FIGURE 12.10 The term “condyloma acuminatum” refers Low-grade squamous intraepithelial lesion to a grossly visible, warty squamous proliferation projecting (LSIL; AIN1) is characterized by the presence of koilocytes above the surrounding mucosa. Condylomata can have quite (A, arrows), which are a hallmark of HPV infection. complex architecture and are now considered to harbor, by Koilocytic changes include hyperchromatic, wrinkled, definition, low-grade squamous dysplasia (AIN1; LSIL). or “raisinoid” nuclei that are surrounded by a sharply demarcated clear cytoplasmic halo. In addition, there is cytologic immaturity and mitotic activity that is limited to the basal third of the epithelium, along with individually (Figure 12.13). In addition, the LAST recommendations dyskeratotic cells (B, arrow). indicate that p16 should be used when the hematoxylin and eosin (H&E) diagnosis is HSIL (P/AIN2), in order to make a more definite diagnosis of either P/AIN3 (in the The characteristic features of koilocytes include nuclear setting of strong, block-like positivity) or LSIL (P/AIN1) “crinkling” or a “raisinoid” appearance, a sharply demar- if the staining is not strong (Figure 12.14). The rationale cated perinuclear clearing or halo and, often, binucleation behind this is the known risk of P/AIN3 as a “precancer- (Figure 12.9A–B). In the past, lesions with koilocytes and ous” lesion associated with high-risk HPV, while LSIL is squamous hyperplasia or acanthosis that appear as grossly visible lesions have been termed “condylomata acumi- nata” (singular, condyloma acuminatum), a moniker still often used clinically. These anogenital “warts” (Figure 12.10) are now classified, according to the LAST project criteria, to harbor low-grade dysplasia, and are included in the category of LSIL. Diagnostic Challenges Glycogenated squamous cells can mimic koilocytes, and inflamed squamous mucosa and/or skin may have reactive atypia that mimics dysplasia (Figure 12.11A–B). Finally, as mentioned in the Introduction, the anal transition zone is lined by a “hybrid” epithelium that is stratified and that can undergo squamous metaplasia. As with metaplasia in the uterine cervix, this can impart an appearance that strongly mimics HSIL (Figure 12.12). The distinction between anal squamous dysplasia FIGURE 12.11 Not every squamous proliferation in and inflammatory and/or metaplastic atypia can be aided the anal canal is a condyloma. In this case, a vaguely using p16 immunohistochemistry as a surrogate marker warty-appearing squamous-lined nodule (A) could be for HPV infection. Strong, block-like p16 positivity should misconstrued as a condyloma, but closer inspection of the push one toward a diagnosis of true dysplasia, while a epithelium (B) reveals only reactive changes with overlying weak, patchy, or negative result suggests reactive atypia hyperkeratosis, suggesting a prolapse-related phenomenon. 12 Neoplasms of the Anus 3 21 FIGURE 12.14 Patchy weak staining with p16 is characteristic of LSIL/AIN1 as in this case, and should prompt downgrading to that diagnosis if one is contemplating a diagnosis of HSIL/AIN2 (courtesy of Keith FIGURE 12.12 The anal transitional mucosa can undergo K. Lai, MD). squamous metaplasia, with loss of the characteristic mucus-producing cells. Like immature squamous metaplasia in the uterine cervix, this appearance can be mistaken for high-grade dysplasia/HSIL. As suggested in the previous paragraph, HSIL lesions are thought to be precursors to invasive squamous cell car- cinoma, although the rate of progression in the anus is con- more likely to be associated with low-risk genotypes; his- troversial. In addition, progression of LSIL to HSIL is also tologic P/AIN2 lesions that are p16 positive are thought thought to be possible, though there is conflicting evidence to behave more like bona fide P/AIN3 lesions. Finally, in the literature. There is some thought that LSIL lesions p16 use is recommended in cases of professional disagree- with block-like p16 positivity may have the highest likeli- ment between observers, when the differential diagnosis hood for progression, though this somewhat belies the LAST includes HSIL. The use of p16 immunostaining is recom- project recommendation that p16 immunohistochemistry be mended against when the histologic findings are thought avoided in lesions thought to be diagnostic of LSIL on H&E to be diagnostic of LSIL (P/AIN1) or HSIL (P/AIN3), as an alone. Therapy for dysplastic squamous lesions in the anus unexpected positive or negative result, respectively, may is similar to that in the genitalia, and centers on eradication simply confuse the issue. of the dysplastic process prior to the development of invasive cancer. Local ablation or surgical excision with the aim of achieving negative margins is standard. INVASIVE SQUAMOUS CELL CARCINOMA Epidemiology and Pathogenesis Squamous cell carcinoma (SCC) is the most common primary anal neoplasm, though it is still relatively rare in comparison to other lower GI neoplasms, accounting for less than 5% of large bowel malignancies. As with the “precancerous” dysplastic lesions discussed in the previ- ous section, most are now believed to be related to under- lying HPV infection, particularly those arising below the dentate line. The risk factors for SCC parallel those for FIGURE 12.13 At very low magnification, the anal squamous dysplasia, including other sexually trans- characteristic block-like p16 positivity of HSIL on the left mitted infections, immunosuppression (including HIV), contrasts with patchy, weak positivity on the right. The and cigarette smoking. In the past, female patients vastly latter

pattern points toward reactive atypia and away from outnumbered males, but this has equalized somewhat in HPV-related dysplasia. recent decades. 3 2 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Clinical and Macroscopic Features occurs in the rectum rather than the anus. Compared to true basal cell carcinomas, basaloid SCCs have more Patients may present with anal pain, with or with- cytologic pleomorphism and often have areas with more out discharge; bleeding; pruritus; or a palpable mass. recognizable squamous differentiation such as keratiniza- Lymphadenopathy may be present, reflecting spread to tion. Their heritage can also be confirmed using markers regional nodes. Early lesions are often small and verru- of squamous differentiation such as p63 immunostaining. coid, whereas later lesions may consist of ulcers, a pal- Conventional SCC arises most often distal to the pable nodule, or a large fungating mass. dentate line, although it can sometimes present when it involves the rectal mucosa. As with other conventional Microscopic Features and Variants SCCs, it may vary from well- to poorly differentiated (Figure 12.16A–D), and can extend proximally into the These carcinomas can have a variety of morphologies, rectum and/or make large, obstructive, ulcerating masses. including large and very well-differentiated verrucous In poorly differentiated tumors, finding foci of keratini- carcinomas (formerly known as “giant condyloma of zation, intercellular bridges/desmosomes, and p63 immu- Buschke–Lowenstein”), basaloid squamous carcinomas nohistochemistry can be helpful in recognizing squamous (referred to as “cloacogenic carcinoma” in the past), and differentiation. typical, keratinizing squamous cell carcinomas identical to Verrucous SCC is typically macroscopically impres- those seen elsewhere in the GI tract and on the skin. sive, reflected in its older name of “giant condyloma.” This Basaloid SCC often arises proximal to the dentate line, name also reflects the difficulty in recognizing that verru- and can be difficult to recognize as a squamous-derived cous tumors are truly invasive, due to their wide, pushing tumor based on H&E histology alone. These tumors con- front rather than infiltrating borders. These tumors can be sist of nests of basophilic cells with relatively scant cyto- very large and, although they are also HPV-related, fea- plasm and peripheral palisading (Figure 12.15A–B). This tures of both viral infection and dysplasia are typically type of carcinoma can mimic the much rarer basal cell difficult to find histologically (Figure 12.17A–B), which carcinoma (arising in the perianal skin) as well as neu- also adds to the difficulty in diagnosing malignancy. roendocrine carcinomas, another rare tumor that usually These tumors often have extensive surface maturation, FIGURE 12.15 Basaloid squamous cell carcinoma of the anus has an appearance at low magnification (A) that strongly resembles basal cell carcinoma (BCC) of the skin, with nests of hyperchromatic cells that have peripheral palisading. At higher magnification (B), however, the cells are more pleomorphic than true BCC, often with brisk mitotic activity. These tumors can also resemble high-grade neuroendocrine carcinomas that may extend from the rectum to involve the anal canal. 12 Neoplasms of the Anus 3 2 3 FIGURE 12.16 Conventional anal squamous cell carcinoma, almost always arising in the setting of HPV infection, varies from well-differentiated (A) with cells that have abundant, pale eosinophilic cytoplasm, to very poorly differentiated (B) with cells that are essentially unrecognizable without ancillary studies such as p63 immunostaining. Intercellular bridges (desmosomes) and keratinization (C) are good morphologic clues to squamous differentiation. Some examples (D) are quite pleomorphic, yet can have areas of keratinization (arrow). and superficial biopsies may only sample benign-appear- ing, hyperparakeratotic epithelium, leading to a benign diagnosis that is at odds with the clinically impressive and worrisome appearance. While the tumor invasion is broad and pushing rather than infiltrative, verrucous SCC are aggressive neoplasms, invading local structures and some- times involving the deep soft tissues of the pelvis. There is some evidence that these deceivingly well-differentiated carcinomas are more closely related to low-risk HPV gen- otypes, in contrast to conventional SCC. Staging Regardless of subtype, anal SCC is staged according to the conventions of the AJCC/UICC TNM manual, sev- enth edition, which currently uses tumor size as the main criterion. The staging scheme is summarized in brief: FIGURE 12.17 Verrucous carcinomas of the anus are very bulky, deceptively well-differentiated masses that can grow • pTx: primary tumor cannot be assessed to be very large (A). As in other sites, the determination of • pT0: no evidence of primary tumor malignancy in such lesions can be difficult, particularly if • pTis: carcinoma in situ (includes HSIL and AIN23) only the superficial part of the process is sampled. Such • pT1: tumor 2 cm or less in the greatest dimension tumors, however, have a broad, pushing invasive front (B) • pT2: tumor more than 2 cm but not more than 5 cm and can be very locally destructive, as in this tumor that in the greatest dimension invades into muscle. • pT3: tumor more than 5 cm 3 2 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide • pT4: tumor of any size that invades adjacent organs the glandular epithelium of the perianal glands/ducts. (eg, vagina, urethra, bladder). Of note, direct invasion Assessment of risk factors and etiologic issues is difficult of rectal wall, perirectal skin, subcutaneous tissues, or given the rarity of these cancers, but there is some evi- anal sphincter muscles is not classified as a T4 lesion. dence associating anal (and even distal colorectal) adeno- Tumors arising above the dentate line tend to metas- carcinomas with HPV. tasize to pelvic, perirectal, and para-aortic/paravertebral lymph nodes, while those arising distally metastasize to Clinical and Macroscopic Features inguinal nodes. Metastasis in perirectal lymph nodes is These tumors typically present in older adults (60–70 classified as N1, whereas metastasis in unilateral inter- years of age), and there is a male predominance. Patients nal iliac and/or inguinal lymph nodes is classified as N2. often have a painful buttock mass and/or a mucinous Metastasis in perirectal and inguinal nodes and/or bilat- anal discharge, and bleeding is less frequently a present- eral internal iliac and/or inguinal nodes is classified as N3. ing symptom than in squamous cell carcinoma. These Current therapy centers on chemoradiation, with radical tumors are aggressive, and patients are at high risk for (abdominoperineal) resection generally reserved for cases both local and distant recurrences; the overall 5-year sur- that fail this therapeutic approach. In general, anal SCC vival rate is 30%. Prognosis is stage dependent, however, has a better prognosis than other primary anal tumors. and staging is similar to anal squamous cell carcinoma. Macroscopically, anal adenocarcinomas arise within ANAL ADENOCARCINOMA the deep soft tissues of the anal region, and intraluminal anal canal growth is rare. Epidemiology and Pathogenesis Primary anal adenocarcinoma is very rare. When an Microscopic Features adenocarcinoma is found to involve the anal canal, the Primary anal gland/duct carcinoma has been described as most likely explanation is distal extension from a primary having one of two morphologic patterns. The first is an rectal tumor, which is the main differential diagnosis for infiltrative process composed of small, angulated tubules primary anal adenocarcinoma. Potential sites for primary lined by malignant epithelial cells with very little luminal anal adenocarcinoma to arise include the mucus-produc- material (Figure 12.18A–B). Alternatively, these carcino- ing epithelial cells of the anal transition zone, as well as mas may have a “colloid” appearance, with clusters of FIGURE 12.18 One form of anal gland/duct carcinoma is characterized by predominantly small, angulated tubules that contain very little luminal material. At low magnification (A), these can be seen infiltrating beneath the overlying squamous epithelium. At high magnification (B), the neoplastic tubules are lined by pleomorphic cells and contain only scant luminal debris. 12 Neoplasms of the Anus 3 2 5 FIGURE 12.19 Another morphologic pattern of anal gland/duct adenocarcinoma consists of large, irregular tubules and/ or paucicellular mucin pools, the so-called “colloid” pattern (A). The neoplastic cells can be reminiscent of colorectal cancer (B) and/or have a signet ring pattern (C). malignant cells associated with large pools of paucicellu- lar mucin (Figure 12.19A–C). Diagnostic Challenges Anal gland/duct carcinomas have an immunophenotype that helps distinguish them from primary rectal tumors that invade the anus. Whereas rectal tumors tend to express cytokeratin (CK)20 and CDX2, primary anal ade- nocarcinoma expresses CK7 and is usually CDX2 nega- tive (Figure 12.20). Additionally, anal adenocarcinomas express MUC5AC, whereas rectal tumors are more likely to express the intestinal mucin phenotype of MUC2. Primary anal adenocarcinoma may have associated individual invasive cells within the squamous epithelium of the distal anal canal and the perianal skin, termed “paget- oid” spread (Figure 12.21). It shares this feature with rectal FIGURE 12.20 Anal gland/duct carcinoma can usually carcinomas, and both are part of the differential diagnosis be distinguished from colorectal carcinoma by its of so-called “(peri)anal Paget disease,” which will be dis- immunostaining pattern, which is characterized by diffuse cussed in the next section. The prognosis of primary anal CK7 positivity (illustrated here), with negativity for CK20 adenocarcinoma is quite poor, and therapy centers on neo- and CDX2. Colonic adenocarcinoma typically has the adjuvant chemoradiation followed by resection. opposite pattern. 3 2 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide FIGURE 12.21 Anal gland/duct adenocarcinoma is one (rare) cause of pagetoid tumor cell spread into the overlying anal squamous epithelium. In this high- magnification view, individual malignant cells, many with mucin vacuoles (arrows), are scattered throughout the FIGURE 12.22 Perianal Paget disease, in either its squamous epithelium overlying a carcinoma. primary or secondary form, is characterized by individually infiltrating malignant cells in the anal squamous mucosa. At low magnification, a large number of these pale- staining cells can be seen within the darker-staining squamous epithelium in this case. (PERI)ANAL PAGET DISEASE Etiology that infiltrate the squamous epithelium of the anal canal Paget disease involving the anus can present a diagnos- and/or perianal skin, termed “pagetoid spread” (Figure tic challenge. It is important to recognize that the histo- 12.22). The cells have a frankly malignant appearance, logic features diagnostic of this entity can arise either as with ample pale cytoplasm, and often contain recogniz- an anal primary (“primary anal/perianal Paget disease”) able mucin vacuoles (Figure 12.23A–B). Some may have or from neoplasms that are centered in the rectum, from the appearance of signet ring cells, and mucin-containing which individual cells infiltrate distally to involve the examples can be highlighted using cytochemical stains anal mucosa (“secondary anal/perianal Paget disease”). such as mucicarmine and/or immunohistochemical mark- Primary Paget disease is much less common than the ers of mucin production. The infiltrative nature of the secondary form, reflecting the common occurrence of process often incites reactive changes in the adjacent squa- colorectal adenocarcinoma in general. The primary form mous epithelium, including hyperparakeratosis, acantho- is similar to that seen in the vulva and perineum, though it sis, and basal atypia. In addition to individual cells, small can occur in both men and women. Primary Paget disease clusters and even tubular structures may be found among is believed to arise from neoplastic cells of either apocrine the squamous cells (Figure 12.24). Macroscopically, the or eccrine origin. involved mucosa and skin may be erythematous, excori- ated, or ulcerated. Identification of Paget disease, as well as the differ- Clinical and Macroscopic Features ential diagnosis between the two forms, can be aided This disease affects both men and women, and typically by immunohistochemistry. It is important to distinguish occurs in patients in the fifth to eight decades. Patients typ- between the two types of Paget disease, because secondary ically present with pruritus and bleeding. Typical lesions Paget disease may be the presenting sign of a rectal neo- consist of crusty, scaly, or ulcerated patches located any- plasm and its identification must prompt a clinical search where between the dentate line and perianal skin. for the underlying carcinoma. As expected, secondary Paget disease has an immunophenotype consistent with colonic adenocarcinoma, expressing CK20, CDX2, and Microscopic Features MUC2 (Figure 12.25A–B); as with other carcinomas of In either of its forms, Paget disease involving the anus the distal colon, there may be some CK7 positivity, too. In is characterized by scattered individual epithelioid cells contrast, primary Paget disease has a phenotype identical 12 Neoplasms of the Anus 3 27 FIGURE 12.23 Perianal Paget disease is characterized FIGURE 12.25 Secondary perianal Paget by cells with ample, pale cytoplasm and a frankly disease most often results from distal extension malignant appearance (A).

Mitotic activity is often of a rectal adenocarcinoma and shares the typical apparent (arrow, A). The malignant cells may also immunophenotype of colon cancer, with CK20 (A) and contain mucin vacuoles (arrow, B), indicative of their CDX2 (B) positivity. adenocarcinoma phenotype. to that seen in cases involving the vulva, which is much Diagnostic Challenges more analogous to mammary carcinoma. Specifically, In addition to the two types of Paget disease, cells from CK7 (Figure 12.26) and GCDFP-15 are positive, as is other tumors arising in and around the anus may spread MUC5AC, while CK20 and CDX2 are negative. GCDFP- in a pagetoid fashion. Specifically, adenocarcinomas from 15 (for “gross cystic disease fluid protein”) is a marker the anal glands/ducts, discussed earlier, and melanomas, of apocrine differentiation that is also expressed by many discussed in the next section, may have this appearance breast carcinomas, and may suggest an apocrine origin for in the squamous epithelium adjacent to the main tumor. Paget disease as well. The immunophenotype of anal gland/duct carcinoma FIGURE 12.24 In addition to individual infiltration, the FIGURE 12.26 Primary perianal Paget disease has a malignant cells of perianal Paget disease may form small unique immunophenotype that is most analogous to that clusters or even tubular structures, which can also contain of breast carcinoma. Such cases are positive for CK7, mucin. illustrated here, and the mammary marker GCDFP-15. 3 2 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide has overlap with that of primary Paget disease, so an index of suspicion and correlation with clinical findings such as an underlying mass are necessary to differenti- ate between these diagnoses. In the case of melanoma, the pagetoid cells contain no mucin and express typi- cal melanocyte markers such as S100, HMB-45, and/or melan-A. Even squamous cell carcinomas may occasion- ally send individual malignant cells into surrounding epithelium in a pagetoid fashion. These express markers of squamous differentiation such as p63, and p16 may be positive in cases associated with HPV. The therapy and prognosis of Paget disease of the anus depends on the specific type. For primary cases, local therapy is aimed at wide excision, though the condi- tion has a propensity to recur and achievement of negative margins can be problematic. Extensive primary cases or those associated with an anal gland/duct carcinoma may FIGURE 12.27 While most melanomas in the GI tract require abdominoperineal resection. are metastatic, primary malignant melanomas can occur Secondary cases with underlying rectal cancer must in the anus. These tumors are morphologically similar to be treated with a plan centered on that primary diagnosis. those seen in the skin, with sheets of very pleomorphic Prognosis in these latter cases depends on the stage of the malignant cells with variably present pigment (A). At high underlying adenocarcinoma, but is usually poor. magnification (B), the typical “dusty” cytoplasm and prominent macronucleoli (arrow) are visible. Pigment is variably present. MELANOMA Epidemiology sarcomatoid variants. These can be more difficult to rec- Primary anal melanoma is quite rare, even in comparison ognize, but S100 expression remains fairly sensitive even to some of the other tumors discussed here, and accounts when other typical melanoma markers are negative. As dis- for only 1% to 3% of all melanomas. Normal melanocytes cussed in the previous section, pagetoid spread by malig- do exist in the anal squamous mucosa and the anal transi- nant melanocytes (Figure 12.29) can be confused with bona tion zone, and are thought to be the precursors to these fide Paget disease, but immunohistochemistry is helpful in tumors. Primary anal melanoma tends to center on the recognizing these cases for what they are. region of the dentate line and, like other tumors discussed in this chapter, can mimic hemorrhoids or anal tags. Thus, it is fairly common for these malignancies to present at a high stage due to lack of suspicion for a malignancy. Pathologic Features Anal melanoma typically manifests as a polypoid mass. Pigment is variably present. The overlying mucosa is often ulcerated, and tumors are often large at presentation. Anal melanomas may occur in the anal canal, at the anal verge, or in the rectum. Anal melanoma is histologically identical to its coun- terparts in the skin, with characteristic collections of large, epithelioid, and very pleomorphic cells (Figure 12.27A–B). Pigment may or may not be present and, given the relatively unusual location, simply including melanoma in the differ- ential diagnosis can be half the battle. Once the diagno- sis is considered, using S100, HMB-45, and/or melan-A to confirm the diagnosis is usually relatively straightforward FIGURE 12.28 As in other sites, melanocytic markers (Figure 12.28). There are reports of other subtypes of mela- such as melan-A (MART-1) highlight the malignant noma occurring in the anus as well, including desmoplastic/ melanoma cells. 12 Neoplasms of the Anus 3 2 9 FIGURE 12.29 Melanoma can also exhibit pagetoid FIGURE 12.30 The perianal skin can give rise to basal spread into the anal squamous epithelium. Here, large cell carcinomas identical to their counterparts elsewhere cells with ample cytoplasm and prominent nucleoli (arrow) in the skin. At low magnification, this carcinoma has the are seen among the squamous cells. At the bottom of the characteristic palisading of cells at the periphery of the figure, some pigment gives a hint as to the nature of these malignant nests, and can be seen to emanate from the cells, but they are otherwise difficult to distinguish from overlying epidermis in one focus (arrow). true perianal Paget disease without immunohistochemistry. The prognosis for anal melanoma is poor, even in cases tend to have less pleomorphism and less mitotic activity. that seem clinically amenable to resection. Furthermore, Immunohistochemistry for EPCAM/BerEp4 can be of radical attempts at resection such as abdominoperineal further help in distinguishing the two, as it tends to be resection do not appear to improve clinical outcomes. positive in BCC and negative in basaloid squamous cell While some cutaneous melanomas have been found to har- carcinoma. Wide excision is the therapy of choice. bor KIT mutations that allow therapy with tyrosine kinase inhibitors, the evidence in anal cases is sparse at this point. BASAL CELL CARCINOMA Basal cell carcinoma (BCC), a cutaneous neoplasm that is rarely encountered in perianal skin, is primarily pre- sented here because it can resemble some of the other entities previously discussed in the section on squamous cell carcinoma. Some patients who develop perianal BCC have a genetic predilection such as the basal cell nevus (Gorlin) syndrome or xeroderma pigmentosum. These tumors develop at or below the anal verge in hair-bearing skin, in contrast to basaloid squamous cell carcinomas that involve the anal canal itself. As in the skin elsewhere, BCCs have a nodular, pearly gross appearance, and can be misidentified as anal tags or hemorrhoids in the clini- cal setting. Histologically, they are composed of nodules of basaloid cells with prominent peripheral palisading, FIGURE 12.31 Most basal cell carcinomas are relatively and they can sometimes be seen “budding” from the basal easy to recognize and to distinguish from basaloid surface of the epidermis (Figure 12.30). Retraction arti- squamous cell carcinomas, because they tend to be less fact may be prominent around the neoplastic nests, and pleomorphic. Potentially confusing histologic features there may be microcystic spaces filled with mucoid mate- in basal cell carcinoma include microcystic spaces that rial or foci of abrupt keratinization (Figure 12.31A–B). In can contain mucoid material (A), and foci of abrupt contrast to basaloid squamous cell carcinoma, true BCCs keratinization (B) that suggest follicular differentiation. 3 3 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide OTHER UNUSUAL ENTITIES College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med. 2012;136(10):1266–1297. In addition to basal cell carcinoma of the perianal skin, Maniar KP, Nayar R. HPV-related squamous neoplasia of the lower other tumors arising in dermal appendages and mesenchy- anogenital tract: an update and review of recent guidelines. Adv mal structures can affect the perianal region. These are Anat Pathol. 2014; 21(5):341–358. very rare, and include apocrine adenocarcinomas, Merkel Pirog EC, Quint KD, Yantiss RK. P16/CDKN2A and Ki-67 enhance the detection of anal intraepithelial neoplasia and condyloma and cor- cell carcinomas, smooth muscle neoplasms, granular cell relate with human papillomavirus detection by polymerase chain tumors, and others. Analogous to primary perianal Paget reaction. Am J Surg Pathol. 2010;34:1449–1455. disease, other mammary analog tumors such as sclerosing Rousseau DL Jr., Thomas CR Jr., Petrelli NJ, Kahlenberg MS. Squamous adenosis-like proliferations can appear in this area as well. cell carcinoma of the anal canal. Surg Oncol. 2005;14:121–132. Smyczek P, Singh AE, Romanowski B. Anal intraepithelial neoplasia: Finally, similar to other apocrine-gland-containing areas, a review and recommendations for screening and management. the perianal region can suffer the effects of hidradenitis Int J STD AIDS. 2013;24:843–851. suppurativa, which produce a mass lesion in some cases. Wong AK, Chan RC, Aggarwal N, et al. human papillomavirus gen- otypes in anal intraepithelial neoplasia and anal carcinoma as detected in tissue biopsies. Mod Pathol. 2010;23:144–150. SELECTED REFERENCES Anal Adenocarcinoma General Jensen SL, Shokouh-Amiri MH, Hagen K, et al. Adenocarcinoma of the anal ducts. A series of 21 cases. Dis Colon Rect. 1988;31:268–272. Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. WHO Meriden Z, Montgomery EA. Anal duct carcinoma: a report of 5 cases. Classification of Tumours of the Digestive System. 4th ed. Lyon, Hum Pathol. 2012;43:216–20. France: IARC Press, 2008. Fargo MV, Latimer KM. Evaluation and management of common ano- rectal conditions. Am Fam Physician. 2012;85:624–630. Paget Disease of the Anus Flejou JF. An update on anal neoplasia. Histopathology. 2015;66(1): 147–160. De Nisi MC, D’Amuri A, Toscano M, et al. Usefullness of CDX2 in the Kazakov D, Spagnolo DV, Kacerovska D, Michal M. Lesions of ano- diagnosis of extramammary Paget disease associated with malig- genital mammary-like glands: an update. Adv Anat Pathol. nancies of intestinal type. Br J Dermatol. 2005;153;677–679. 2011;18:1–28. Goldblum JR, Hart WR. Perianal Paget’s disease: a histologic and Klein JW. Common anal problems. Med Clin North Am. immunohistochemical study of 11 cases with and without associ- 2014;98(3):609–623. ated rectal adenocarcinoma. Am J Surg Pathol. 1998;22:170–179. Leonard D, Beddy D, Dozois EJ. Neoplasms of anal canal and perianal Regauer S. Extramammary Paget’s disease: a proliferation of adnexal skin. Clin Colon Rectal Surg. 2011;24(1):54–63. orgin? Histopathology. 2006;48:723–729. Longacre TA, Kong CS, Welton ML. Diagnostic problems in anal Tulchinsky H, Zmora O, Brazowski E, et al. Extramammary Paget’s pathology. Adv Anat Pathol. 2008;15:263–278. disease of the perianal region. Colorectal Dis. 2004;6:206–209. Shia J. An update on tumors of the anal canal. Arch Pathol Lab Med. 2010;134(11):1601–1611. Primary Anal Melanoma Felz MW, Winburn GB, Kallab AM, Lee JR. Anal melanoma: an Infl ammatory/Non-Neoplastic Lesions aggressive malignancy masquerading as hemorrhoids. South Med Chetty R, Rhathal PS, Slavin JL. Prolapse-induced inflammatory pol- J. 2001;94:880–885. yps of the colorectum and anal transition zone. Histopathology. Heyn J, Placzek M, Ozimek A, et al. Malignant melanoma of the anal 1993;23:63–67. region. Clin Exp Dermatol. 2007;32:603–607. Ganz RA. The evaluation and treatment of hemorrhoids: a guide for the Kanaan Z, Mulhall A, Mahid S, et al. A systematic review of prognosis gastroenterologist. Clin Gastroenterol Hepatol. 2013;11:593–603. and therapy of anal malignant melanoma: a plea for more precise Mathialagan R, Turner MJ, Gorard DA. Inflammatory cloacogenic reporting of location and thickness. Am Surg. 2012;78:28–35. polyp mimicking anorectal malignancy. Eur J Gastroenterol Hepatol. 2000;12:247–250. Perianal Basal Cell Carcinoma Squamous Dysplasia and Carcinoma Nagendra Naidu DV, Rajakumar V. Perianal basal cell carcinoma-an unusual site of occurrence. Indian J Dermatol. 2010;55:178–180. Daling JR, Madeleine MM, Johnson LG, et al. Human papillomavi- Patil DT, Goldblum JR, Billings SD. Clinicopathologic analysis of basal rus, smoking, and sexual practices in the etiology of anal cancer. cell carcinoma of the anal region and its distinction from basaloid Cancer. 2004;101:270–280. squamous cell carcinoma. Mod Pathol. 2013;26:1382–1389. Darragh TM, Colgan TJ, Cox JT, et al. The lower anogenital squa- Wang SQ, Goldberg LH. Multiple polypoid basal cell carcinomas on mous terminology standardization project for HPV-associated the perineum of a patient with basal cell nevus syndrome. J Am lesions: background and consensus recommendations from the Acad Dermtol. 2007;57(Suppl 2): S36–S37. 13 Applications of Diagnostic Immunohistochemistry ANDREW M. BELLIZZI INTRODUCTION type. When initially evaluating a malignant neoplasm of uncertain type, a useful immunohistochemical screening It is fair to say that as of today no special technique has influ- panel includes

a broad-spectrum keratin, S100, and leu- enced the way that pathology is practiced as profoundly as kocyte common antigen (LCA; also known as CD45), to immunohistochemistry, or has come even close to it. recognize carcinoma, melanoma, and hematolymphoid —Juan Rosai neoplasms, respectively. Keratins represent the principal structural protein of The ability to visualize antigen expression in the context epithelia. Fifty-four human keratins have been described, of tissue morphology is incredibly powerful, allowing us with most epithelia expressing keratins 4 to 8. Stratified to literally differentiate the undifferentiated. In addition epithelia also highly express keratins 1 to 6 and 9 to 17, to familiar diagnostic applications including diagnosis of while simple epithelia express combinations of keratins 7, broad tumor class, discernment of carcinoma type, and 8, 18, 19, and 20, among others. Broad-spectrum kera- determination of site of origin, immunohistochemistry tin immunostains recognize multiple keratins, and com- (IHC) applications are increasingly prognostic and pre- mercially available clones including AE1/AE3, OSCAR, dictive. Next-generation IHC takes advantage of discov- MAK-6, MNF116, and CAM5.2 are helpful in diagnos- eries in developmental biology and molecular genetics; ing poorly differentiated carcinomas. Other broad-spec- mining of that literature has revealed lineage-restricted trum epithelial markers include monoclonal antibodies to transcription factors, biomarkers identified by gene EPCAM (eg, MOC-31, BerEp4), MUC1 (more commonly expression profiling, and protein correlates of molecular referred to as EMA), and claudin-4. As a note of cau- genetic events. This chapter will follow the outline of this tion, broad-spectrum epithelial markers are occasionally entire book, proceeding from more general, approach- expressed by nonepithelial tumors including select sarco- oriented applications to organ-specific ones. mas (especially epithelioid sarcomas), hematolymphoid neoplasms (especially anaplastic large cell lymphoma and plasma cell neoplasms), melanomas (up to 20% of meta- APPROACH TO EPITHELIAL NEOPLASMS static tumors), and, of course, mesothelioma (keratin and EMA-positive, while MOC-31 and claudin-4-negative). Immunohistochemistry often plays a critical role in defin- S100 expression is very sensitive for a diagnosis of ing tumor type, especially in poorly differentiated neo- melanoma, but the protein is fairly widely expressed (eg, plasms, and helps assign the primary site of origin in Schwann cells, adipocytes, chondrocytes, dendritic cells, tumors presenting as metastases. In the gastrointestinal Langerhans cells, myoepithelial cells), and positivity has (GI) tract, it may be challenging to distinguish an adeno- been reported in over a third of adenocarcinomas. The carcinoma of GI origin from a metastasis or another tumor melanoma markers melan-A and HMB-45 are more 3 31 3 3 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide specific, though less sensitive. The transcription factor GATA3 is highly expressed by urothelial carcinoma; it is SOX10, combining superior sensitivity and specificity, is also occasionally expressed by squamous cell carcinoma, becoming more widely used diagnostically; in addition to in particular those of genitourinary or cutaneous origin, melanoma, it is expressed by nerve sheath and myoepithe- although expression in squamous cell carcinoma is typi- lial tumors and some gliomas. cally weaker than that seen in urothelial carcinoma. Among the “big three” of keratin, S100, and LCA, LCA Carcinomas composed of large polygonal cells give is the most specific, although some lymphoid neoplasms are rise to a different set of diagnostic considerations, includ- negative for LCA (eg, plasmablastic lymphoma and some ing hepatocellular carcinoma (HCC), renal cell carcinoma anaplastic large cell lymphomas). For this reason, it is rea- (RCC), and adrenal cortical carcinoma. These three tumor sonable to add immunostains for CD79a, MUM1, ALK, types are typically negative for both CK7 and CK20. Key and CD30 in keratin/S100/LCA “triple-negative” tumors diagnostic markers for these tumors include Hep Par 1 and in which a hematolymphoid neoplasm remains a diagnostic glypican-3 (HCC); PAX8 (RCC); and melan-A, inhibin, consideration morphologically. and, more recently, the transcription factor steroidogenic There is no broad-spectrum marker specific for mes- factor 1 (SF1) for adrenal cortical carcinoma. Of note, it is enchymal tumors. Although vimentin is widely utilized as the melan-A clone A103 that reacts with adrenal cortical such a marker, it is also highly expressed by melanoma, carcinoma, while other clones (eg, M2-7C10) may only lymphoma, and some carcinomas (eg, endometrial and react with melanoma. Adrenal cortical carcinoma also kidney, as well as sarcomatoid carcinomas from any site). frequently expresses synaptophysin (50%–75%), (but not CD34 is often expressed by sarcomas, especially vascular chromogranin), which may lead to an incorrect diagnosis and fibroblastic tumors, but also occasionally by less dif- of a neuroendocrine tumor (NET). ferentiated tumors. As it is only exceptionally expressed by The neuroendocrine neoplasms occurring in the GI carcinomas, the demonstration of CD34 expression in a tract, both primary and metastatic, include well-differ- poorly differentiated malignant neoplasm of uncertain type entiated NETs and poorly differentiated neuroendocrine is useful to reasonably exclude a diagnosis of carcinoma. carcinomas (NECs). The presence of neuroendocrine dif- Broad tumor classes and immunostains useful in the ferentiation can be confirmed with the general neuroendo- diagnosis of poorly differentiated examples are summa- crine markers chromogranin A and synaptophysin, while rized in Table 13.1. the Ki-67 proliferation index is useful for assigning grade (see also Chapter 3). An approach to the distinction of car- Primary Versus Metastatic cinoma types is presented in Algorithm 13.1. Carcinomas in the GI Tract Well- and moderately differentiated adenocarcinomas are usually readily recognized based on gland/papillae for- Squamous cell carcinomas typically produce keratin, and mation and/or mucin production. However, the distinc- have demonstrable intercellular bridges corresponding to tion between primary and metastatic adenocarcinomas in desmosomes. p63 and cytokeratin (CK)5/6 are highly sen- the GI tract may be challenging. Patterns of coordinate sitive and moderately specific for the squamous and tran- CK7 and CK20 expression (see Table 13.2), supplemented sitional cell carcinomas (Figure 13.1A–D) as well as for by more specific differentiation markers (see Table 13.3), metastatic squamous cell carcinoma. are useful in assigning the site of origin in adenocarcino- Urothelial (transitional cell) carcinomas have con- mas as well as the large polygonal cell tumors mentioned siderable morphologic overlap with squamous cell in the preceding paragraphs (Figure 13.2A–E). Overall, carcinomas, and may show foci of overt squamous dif- most adenocarcinomas are CK7+/CK20-; urothelial car- ferentiation. Approximately half of urothelial carcinomas cinomas, upper GI tract adenocarcinomas, and mucinous coexpress CK7/CK20, while squamous cell carcinomas ovarian neoplasms are often CK7/CK20 “double posi- are CK7 variable/CK20-negative. The transcription factor tive;” and colon and Merkel cell carcinomas are typically TABLE 13.1 Useful Immunostains in Poorly Differentiated Malignant Neoplasm Tumor Type Useful Immunostains Carcinoma Broad-spectrum keratins (eg, AE1/AE3, OSCAR, MAK-6, MNF116, CAM 5.2), EMA Melanoma S100, melan-A/MART-1, HMB-45, MiTF, SOX10 Hematolymphoid neoplasm CD45/LCA; (CD43, CD79a, MUM1, ALK, CD30 if LCA-negative) Sarcoma CD34; MDM2/CDK4 (dedifferentiated liposarcoma); additional based on morphology Mesothelioma WT-1 (nuclear), calretinin, CK5/6, D2-40 Germ cell tumor SALL4, PLAP Neuroendocrine Chromogranin, synaptophysin 13 Applications of Diagnostic Immunohistochemistry 3 3 3 (A) (B) (C) (C) (D) FIGURE 13.1 This essentially undifferentiated anal canal tumor has a syncytial quality and contains frequent tumor infiltrating lymphocytes (A). p63 is diffusely, strongly expressed, supporting a diagnosis of squamous cell carcinoma (B). CDX2 is not expressed (C). p16 demonstrates diffuse, strong expression, suggesting an etiologic association with high-risk human papillomavirus (see later discussion) (D). This squamous cell carcinoma variant has been referred to as lymphoepithelioma-like carcinoma. Carcinoma Adenocarcinoma Squamotransitional (gland forming/ Large polygonal cell Neuroendocrine (p63, CK5/6+) (CG, SYN+) mucin producing)— see next three tables NET NEC Ki-67 Ki-67 ≤20% >20% SCC HCC CK7 var. Hep Par 1+ GPC3+ AdCC GATA3-/+ UC RCC melan-A+ CK7/CK20+ PAX8+ inhibin+ GATA3+ SYN+ ALGORITHM 13.1 Immunohistochemical Approach to Carcinoma Type. Abbreviations: AdCC, adrenal cortical carcinoma; CG, chromogranin; GPC3, glypican-3; HCC, hepatocellular carcinoma; NEC, neuroendocrine carcinoma; NET, neuroendocrine tumor; RCC, renal cell carcinoma; SCC, squamous cell carcinoma; SYN, synaptophysin; UC, urothelial carcinoma. 3 3 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 13.2 Broad Patterns of CK7/CK20 various patterns of CK7/CK20 coordinate expression in Coordinate Expression the GI tract are presented in Table 13.4. There is also variation in CDX2 expression in the GI Site CK7 CK20 tract. Most upper tubal gut adenocarcinomas are CDX2- positive (up to 80%), though expression tends to be weak Prostate, HCC, RCC, AdCC − − Lung (adenocarcinoma), breast, Müllerian, + − to moderate and somewhat patchy (ie, heterogeneous) upper GI, pancreatobiliary (Figure 13.3A–B). CDX2 is expressed by 20% of pancrea- Bladder (UC), upper GI, pancreatobiliary, + + tobiliary tract adenocarcinomas, again typically in a het- mucinous ovarian erogeneous fashion. In colon cancer, expression is usually Colon, Merkel cell carcinoma − + diffuse and strong (ie, homogeneous) (Figure 13.3C–D). As mentioned in the preceding paragraphs, MSI-H tumors are more likely to be CK20-negative than microsatellite stable (MSS) tumors, and CDX2 may also be weak to negative in CK7-/CK20+. In addition, as noted previously, HCC, this circumstance. Abnormal mismatch repair (MMR) pro- RCC, and adrenal cortical carcinoma, along with prostate tein IHC may be useful in this setting to secure the diagno- cancer, are CK7/CK20 “double negative.” Transcription sis (Figure 13.4). factors are increasingly utilized as differentiation markers In the ovary, metastatic GI adenocarcinoma must be for the site of origin as well. CDX2 is a key GI differen- distinguished from mucinous tumors of ovarian origin. tiation marker, and other emerging markers of intestinal Mucinous ovarian neoplasms are generally strongly CK7- differentiation include SATB2 and CDH17. positive, while CK20 and CDX2, when expressed, tend to There is greater diversity of CK7/CK20 expression in be heterogeneous. This phenotype ov erlaps with that seen GI tract adenocarcinomas than is generally appreciated. in the upper GI tract and pancreatobiliary adenocarcino- Esophageal adenocarcinomas are most commonly CK7+/ mas. Loss of SMAD4 expression supports a pancreatic CK20-; gastric cancers are fairly evenly split between the primary (seen in 50%), while PAX8 expression supports four combinations of CK7/CK20 expression; and small an ovarian primary (seen in 20%–40%). Primary disease intestinal carcinomas are more commonly CK7/CK20 tends to be unilateral and quite large (ie, greater than 13 “double positive.” Pancreatobiliary adenocarcinomas are cm), while metastatic disease is more likely to be bilateral. nearly always strongly CK7-positive, and 40% to 60% are CK20-positive, though the positivity often manifests as only rare immunoreactive cells. Most colon cancers are APPROACH TO NEUROENDOCRINE CK7-/CK20+, though about 10% are “double positive” NEOPLASMS and 10% are “double negative.” CK7+/CK20+ tumors are common in the rectum, where they comprise up to Neuroendocrine epithelial neoplasms are characterized 25% of adenocarcinomas. CK20-negative tumors tend by expression of general neuroendocrine markers and to be poorly differentiated and/or demonstrate high-level keratins, as well as production of peptide hormones and/ microsatellite instability (MSI-H). The frequencies of the or biogenic amines. In the current WHO classification, TABLE 13.3 Immunohistochemistry to Assign Adenocarcinoma Site of Origin: Differentiation Markers Marker Specificity Transcription Factor CDX2 Enteric differentiation Yes TTF-1 Lung, thyroid Yes ER Breast, Müllerian Yes PR Breast, Müllerian Yes PAX8 Kidney, Müllerian, thyroid Yes p53 Serous carcinoma Yes WT-1 (nuclear) Serous carcinoma, mesothelioma Yes GATA3 Breast (also urothelial carcinoma, paraganglioma/ Yes pheochromocytoma, choriocarcinoma, yolk sac tumor) Napsin A Lung, papillary renal cell carcinoma, Müllerian clear cell No carcinoma PSA Prostate No PSAP Prostate No Thyroglobulin Thyroid No GCDFP-15 Breast No Mammaglobin Breast No 13 Applications of Diagnostic Immunohistochemistry 3 3 5 (A) (B) (C) (D) (E) FIGURE 13.2 This tumor, which exhibits tubulopapillary architecture, involves the rectum in an “outside-in”/mural-based fashion, sparing the mucosa (A). At higher power the tumor is composed of clear to eosinophilic cells, which “hobnail” and are associated with flocculent eosinophilic secretions (B). The tumor expresses CK7 (C) but not CK20 (D); CDX2 and WT-1 were also negative, while the transcription factor PAX8 (E) is diffusely, strongly expressed. This morphology and immunophenotype support a diagnosis of involvement by Müllerian clear cell carcinoma and argue against a GI primary. 3 3 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 13.4 CK7/CK20 Coordinate Expression in Gastroenteropancreatobiliary Adenocarcinomas CK7−/CK20− CK7+/CK20− CK7+/CK20+ CK7−/CK20+ Esophagus 8% (7/85) 74% (63/85) 15% (13/85) 2% (2/85) Stomach 14% (5/37) 19% (7/37) 32% (27/37) 35% (13/37) Small intestine 0% (0/24) 33% (8/24) 67% (16/24) 0% (0/24) Pancreas 3% (1/36) 28% (10/36) 64% (23/36) 6% (2/36) Biliary tree 7% (1/14) 50% (7/14) 43% (6/14) 0% (0/14) Colon 10% (6/60) 0% (0/60) 8% (5/60) 82% (49/60) Sources: Wang NP, Zee S, Zarbo RJ, Bacchi CE, Gown AM. Coordinate expression of cytokeratins 7 and 20 defined unique subsets of carcino- mas. Appl Immunohistochem. 1995;3(2):99–107; Chu P, Wu E, Weiss LM. Cytokeratin 7 and

cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13(9):962–72; Taniere P, Borghi-Scoazec G, Saurin JC, Lombard-Bohas C, Boulez J, Berger F, et al. Cytokeratin expression in adenocarcinomas of the esophagogastric junction: a comparative study of adenocarcinomas of the distal esophagus and of the proximal stomach. Am J Surg Path. 2002;26(9):1213–1221; Chen ZM, Wang HL. Alteration of cytokeratin 7 and cytokeratin 20 expres- sion profile is uniquely associated with tumorigenesis of primary adenocarcinoma of the small intestine. Am J Surg Path. 2004;28(10):1352–1359. (A) (B) (C) (D) FIGURE 13.3 Weak, patchy CDX2 expression in a primary diffuse-type gastric cancer (A). Somewhat stronger, more diffuse CDX2 positivity in an esophageal adenocarcinoma (B). Diffuse, strong (homogeneous) CDX2 expression is seen in a colonic adenocarcinoma with mucinous features (C) and a rectal signet ring cell adenocarcinoma (D). Staining that is less than diffuse and strong is referred to as heterogeneous, which is more consistent with upper GI, pancreatobiliary, or mucinous ovarian origin. 13 Applications of Diagnostic Immunohistochemistry 3 37 (A) (B) (C) (D) FIGURE 13.4 This tumor in the right colon is composed of cords of large cells with frequent tumor infiltrating lymphocytes (A). The tumor does not express CK7, CK20, or CDX2 (B) and was originally interpreted as a metastasis. MLH1 (C) and PMS2 are absent in tumor cells (note intact staining in intraepithelial lymphocytes), while MSH2 (D) and MSH6 expression are intact. Undifferentiated, microsatellite unstable colon cancer is referred to as medullary carcinoma, and it frequently deviates from the typical CK7-/CK20+/CDX2+ CRC immunophenotype. well-differentiated examples are referred to as neuroendo- carcinomas express TTF-1, while CK20 is rarely expressed. crine tumors (NETs) and poorly differentiated examples Merkel cell carcinoma (primary NEC of the skin) has the as neuroendocrine carcinomas (NECs). IHC has several inverse immunophenotype, and CK20 often shows a char- applications regarding the recognition and classification of acteristic “dot-like” pattern of positivity. Extrapulmonary neuroendocrine epithelial neoplasms (see also Chapter 3), visceral small cell carcinomas often express TTF-1 as well specifically: (40%–50%), while CK20 is again rarely expressed. Aside • Preferred status of chromogranin A and synapto- from these two markers, IHC has a limited role in assign- physin over other markers in determining the pres- ing the site of origin, with NECs frequently expressing ence of neuroendocrine differentiation multiple transcription factors regardless of the site of ori- • Importance of Ki-67 in accurately grading neoplasms gin (so-called “transcription factor infidelity”). • Utility of IHC panels to assign the site of origin of In contrast, NETs demonstrate fairly characteristic neoplasms of unknown origin protein expression patterns, as determined by the site of origin (Figure 13.5). This is useful, as 10% to 20% of NETs In terms of differentiating primary GI NECs from (13% in a recent analysis of Surveillance, Epidemiology, metastases, 90% or greater of pulmonary small cell and End Results data from 35,825 tumors) present as 3 3 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 13.5 This patient presented with synchronous neuroendocrine tumors in the pancreas (A) and ileum (B). The pancreatic tumor expresses ISL1 (C) but not CDX2 (D). The ileal tumor demonstrates the inverse immunophenotype— CDX2 positivity (E) and ISL-1 negativity (F)—supporting the presence of two independent primaries rather than a metastasis. 13 Applications of Diagnostic Immunohistochemistry 3 3 9 metastases of unknown primary site, and medical and CDX2 expression in the absence of PAX6 or Islet 1 expres- surgical therapeutic decision making is heavily influenced sion are assigned a jejunoileal origin, while tumors with by the primary site. TTF-1 expression is specific for a any PAX6 and/or Islet 1 expression are classified as pan- bronchopulmonary origin, although the sensitivity of this creatic in origin (regardless of CDX2 expression, which is marker is only 30% to 40%. Gastric tumors often dem- known to occur at varying intensity in 15% of pancreatic onstrate a “null” transcription factor immunophenotype. NETs). CDX2/PAX6/Islet 1 “triple negative” tumors fur- Pancreatic and duodenal tumors are not readily separable, ther undergo testing for PR, PDX1, NESP55, and PrAP with characteristic markers including polyclonal PAX8, expression. PrAP-positive tumors are of suspected jejuno- monoclonal PAX6, Islet 1, PR, PDX1, and NESP55. Eighty ileal origin, while tumors expressing PR, PDX1, and/or to ninety percent of midgut (ie, jejunoileal and appendi- NESP55 are of suspected pancreatic origin. Four percent ceal) tumors express CDX2, which is typically diffuse of the tumors in this study were “pan-negative” for all 7 and strong; about half express prostatic acid phosphatase markers; interestingly, these tumors were all jejunoileal. (PrAP). Rectal tumors have an overlapping immunophe- notype with pancreaticoduodenal tumors, with frequent expression of polyclonal PAX8, monoclonal PAX6, and APPROACH TO HEMATOLYMPHOID TUMORS Islet 1; they are distinguished by frequent expression of PrAP and SATB2. Of all these anatomic sites, jejunoileal The GI tract is the most common site for extranodal lym- (especially) and pancreatic tumors are most likely to pres- phomas (see also Chapter 4). At least half of these occur in ent as metastases of occult origin. the stomach, with diffuse large B-cell lymphoma (DLBCL) Maxwell and colleagues recently evaluated the useful- and extranodal marginal zone lymphoma of mucosa- ness of an IHC panel in determining NET site of origin. associated lymphoid tissue (MALT lymphoma) occurring Given the epidemiology of metastatic NETs of unknown at similar frequencies. Throughout the entire GI tract, origin, the approach in Algorithm 13.2 was developed to DLBCL accounts for almost half of all lymphomas, with specifically assign a midgut or pancreatic origin. Their MALT lymphoma next most frequent at 20%. The dif- approach was 94% accurate in a set of 123 tumors (86 pri- ferential immunophenotype of the hematolymphoid neo- mary, 37 metastatic). Tumors are first stained with anti- plasms most commonly encountered in the tubal gut is bodies to CDX2, PAX6, and Islet 1. Tumors with strong summarized in Table 13.5. Perform CDX2, PAX6, and Islet 1 TIER 1 CDX2+ (strong); CDX2/PAX6/Islet 1− PAX6 and/or Islet 1+; PAX6 and Islet 1− (“Triple Negative (CDX2-/+) (“Midgut Pattern”): Pattern”): (“Pancreatic Pattern, Jejunoileal 1° Indeterminate “Hybrid Pattern”): Pancreatic 1° Perform PR, PDX1, NESP55, PrAP TIER 2 PR/PDX1/NESP55/PrAP- PR, PDX1, and/or PrAP+: (“Pan-Negative Pattern”): NESP55+: Suspect Jejunoileal 1° Indeterminate Suspect Pancreatic 1° ALGORITHM 13.2 Approach to Assigning Site of Origin in a Neuroendocrine Tumor of Unknown Origin. Abbreviation: PrAP, prostatic acid phosphatase. Source: Maxwell JE, Sherman SK, Stashek KM, O’Dorisio TM, Bellizzi AM, Howe JR. A practical method to determine the site of unknown primary in metastatic neuroendocrine tumors. Surgery. 2014 Dec;156(6):1359–1366. 3 4 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 13.5 Immunophenotype of GI Hematolymphoid Neoplasms Most Common CD20 CD79a Tumor Type Location(s) PAX5 CD10 Bcl-6 CD5 CD43 Cyclin D1 Bcl-2 Other Useful Markers/Notes Tumors Composed of Small Cells Extranodal marginal zone lym- Stomach + − − Occ. (30%) − Var. Kappa/lambda light chain restriction (occ.); phoma of mucosa-associated keratins to highlight lymphoepithelial lymphoid tissues (MALT lesions lymphoma) Mantle cell lymphoma Colon, small intestine + − + + + + Ki-67 proliferation index >40%–60% associated with poor prognosis; rarely CD5 or cyclin D1- Follicular lymphoma Small intestine + + − − − + Higher-grade tumors more likely to show aberrant immunophenotype (eg, CD10- or CD43+) Chronic lymphocytic leukemia/small Peri-tubal gut/ + − + + − + CD23+ lymphocytic lymphoma mesenteric lymph nodes Enteropathy-associated T-cell Small intestine − − − + − + CD3+, CD7+, CD4−, CD8+, CD56+ lymphoma, Type II Tumors Composed of Intermediate/Large Cells Diffuse large B-cell lymphoma Anywhere + CD10 Var. Rare Occ. (25%) − Var. MUM1 (35% –65%) (30%–60%) (10%) Bcl-6+ (60%–90%) Burkitt lymphoma Ileocecal region + + − + − − Ki-67 proliferation index approaches 100%; TdT-; c-Myc+; Bcl-2 occ. weak+ Plasmablastic lymphoma Rectum CD20/ PAX5- + − + − + EBV EBER (60% –75%); CD45-; MUM1/ CD79a+ CD138/ CD38+; EMA/CD30 var.; (50%–85%) association with HIV Granulocytic sarcoma Lymph nodes (in GI − − − + − − CD68/KIT/CD99/ CD34/TdT Var. tract) B-lymphoblastic lymphoma Liver, lymph nodes (in CD20 Var. CD10+ (60%) − + (67%) − + TdT (95%), CD34/CD99+; CD13/CD33 Occ. GI tract) (25%–50%) Bcl-6- CD79a+ PAX5+ T-lymphoblastic lymphoma Liver, lymph nodes (in CD20/ PAX5- CD10 Var. Var. + (90%) − + TdT (95%), CD3/CD99+; CD4/CD8 double+ GI tract) CD79a Rare Bcl-6- (70%); CD1a (67%); CD34 Var.; CD13/ (5%–10%) CD33 Occ. Enteropathy-associated T-cell Small intestine − − − + − + CD3+, CD7+, CD4−, CD8−/+; association lymphoma, Type I with celiac disease Key: Rare, ≤10%; Occ., >10% and <30%; Var. ≥30% and <60%. 13 Applications of Diagnostic Immunohistochemistry 3 41 Pathologists typically encounter GI tract lymphomas high-grade malignancies including carcinoma, melanoma, in two main morphologic contexts. The first is the pres- sarcoma, and germ cell neoplasm. ence of a small lymphocytic infiltrate, which may present When contemplating the differential diagnosis of a diffusely, as an intraepithelial lymphocytosis, and/or as lymphoma composed of small lymphocytes versus an nodules. The second is an infiltrate of atypical intermedi- inflammatory/reactive condition, morphologic features ate to large cells. In the first instance, lymphomas com- favoring lymphoma include effacement and infiltration posed of small lymphocytes must be distinguished from of the normal architecture by the infiltrate, monoto- inflammatory conditions (eg, Helicobacter pylori gastritis nous cytomorphology, and the inability to identify nor- in the stomach) and exuberant responses to physiologic mal germinal centers (Figure 13.6A–B). Screening IHC antigenic stimulation (eg, Peyer’s patches in the ileum). may include the pan-B-cell marker CD20 and the pan- In the second instance, cancer should be strongly sus- T-cell marker CD3 (Figure 13.6C–D). The vast major- pected, and lymphoma must be distinguished from other ity of lymphomas in this setting are B-cell lymphomas, (A) (B) (C) (D) FIGURE 13.6 These mucosal biopsies were taken at screening colonoscopy, in which a few small polyps were found (A). The fragment on the right demonstrates architectural effacement by a diffuse, small-blue-cell process. At higher power, a monotonous infiltrate of small lymphocytes with somewhat irregular nuclear contours is evident (B). CD20 is diffusely, strongly expressed (C), while CD3 highlights scattered T-cells (D), supporting a diagnosis of a low-grade B-cell lymphoma. This tumor was further shown to coexpress CD5 and cyclin D1 and was thus interpreted as mantle cell lymphoma. 3 42 Neoplastic Gastrointestinal Pathology: An Illustrated Guide with the CD20 and CD3 staining revealing “too many useful markers include the germinal center markers CD10 small B-lymphocytes.” CD10 and BCL6 may be useful and BCL6, and the activated B-cell marker MUM1, to highlight germinal center cells, while CD21, CD23, among others. or CD35 may be applied to identify follicular dendritic Burkitt lymphoma is characterized by monomorphous meshworks. cytomorphology, a germinal center phenotype (CD10/ The principal considerations when diagnosing a lym- BCL6+), and a Ki-67 proliferation index approaching phoma composed of small lymphocytes in the GI tract 100% (Figure 13.7). Diffuse, strong nuclear expression of include MALT lymphoma, mantle cell lymphoma (MCL), c-Myc can serve as an IHC surrogate of a MYC translo- follicular lymphoma (FL), and chronic lymphocytic leu- cation. TdT-negativity distinguishes this tumor from lym- kemia/small lymphocytic lymphoma (CLL/SLL) (see also phoblastic lymphoma, and Bcl-2 is usually not expressed Chapter 4). Useful markers in this context, in addition to (and if expressed should not be more than weakly positive). those already mentioned, include the pan-B-cell markers Plasmablastic lymphoma has an association with CD79a and PAX5 (especially in the setting of rituximab- human immunodeficiency virus. Most cases are LCA, treated tumors, in which CD20 expression is often abol- CD20, and PAX5-negative, though CD79a is usually ished), CD5 (expressed by MCL and CLL/SLL), CD43 expressed, as are markers of plasmacytic differentia- (aberrantly coexpressed by MCL, CLL, and up to 30% of tion. In most cases (60% to 75%) Epstein–Barr virus MALT lymphomas), and cyclin D1 (essentially diagnos- (EBV)-encoded RNA (EBER) expression can be detected tic of MCL). Although immunostains for Bcl-2 are often by in situ hybridization. The most consistent markers applied, this marker is most useful in the setting of a nod- of granulocytic sarcoma include LCA and CD43, with ular lymphoid proliferation, where expression favors FL CD68, KIT, CD99, CD34, and TdT variably expressed. over reactive follicular hyperplasia. Other markers may Lymphoblastic lymphomas nearly always express TdT, also be useful in select settings. For example, kappa or with CD99 also consistently expressed, and CD34 lambda light chain restriction (detectable by IHC or in situ more often expressed in B-lymphoblastic

lymphoma hybridization) is occasionally seen in MALT lymphomas than T-lymphoblastic lymphoma. CD79a and PAX5 with plasmacytic differentiation, and keratin staining may are often positive in B-lymphoblastic lymphoma, while be useful to highlight lymphoepithelial lesions in MALT T-lymphoblastic lymphoma is usually CD3-positive, lymphoma. In MCL, Ki-67 IHC may be applied, as higher often CD4/CD8-“double positive,” and CD1a-positive. proliferation indices appear prognostically adverse. CD23 Post-transplant lymphoproliferative disorders also coexpression may be helpful to secure a diagnosis of CLL/ involve the GI tract, as well as lymph nodes, lungs, liver, SLL. The details of the less commonly encountered immu- or the allograft itself (Figure 13.8). Most cases are EBV- nohistochemical diagnosis of T-cell lymphomas in the driven, and thus EBER-positive. The morphologic spec- GI tract are discussed in Chapter 4, but summarized in trum includes polymorphic and monomorphic disorders, Table 13.5. generally of B-cells. Most monomorphic examples resem- When faced with an intermediate to large cell pro- ble DLBCL. cess, the first order of business is to distinguish hemato- lymphoid malignancies from other high-grade tumors. As previously discussed in the approach to epithelial APPROACH TO MESENCHYMAL TUMORS neoplasms section, important screening markers include LCA, a broad-spectrum keratin, and S100. Some high- Mesenchymal tumors involve the tubal gut more than grade lymphomas are typically LCA-negative (eg, plasma- any other visceral organ. Gastrointestinal stromal tumor blastic lymphoma), and when faced with an LCA/keratin/ (GIST) is recognized as the most common mesenchymal S100-negative tumor, application of additional pan-B-cell tumor of the GI tract and should always be considered in (PAX5, CD79a), T-cell (CD3), and plasmacytic markers the differential of both spindle cell and epithelioid tumors. (CD38, CD138, MUM1) should be considered. As a note At the turn of the century, with the discovery of KIT and of caution, CD138 (also known as syndecan-1) is fre- PDGFRA activating mutations, availability of KIT IHC, quently expressed by carcinomas. and recognition of the efficacy of imatinib, awareness of As noted in the preceding paragraphs, most hema- this entity increased rapidly. That being said, there are a tolymphoid neoplasms in the GI tract composed of number of non-GIST GI mesenchymal tumors, several of intermediate to large cells are DLBCLs. Additional diag- which demonstrate significant morphologic and immuno- nostic considerations include Burkitt lymphoma (BL), phenotypic overlap with GIST (see also Chapter 5). The plasmablastic lymphoma, granulocytic sarcoma, B- and differential immunophenotype of GI mesenchymal tumors T-lymphoblastic lymphoma, and, rarely, type I EATL. is summarized in Table 13.6, and some specific examples In DLBCL, IHC may be applied to determine whether a by site are discussed directly below. tumor is of germinal center or activated-B-cell type, as the In the esophagus, leiomyomas are the most com- latter designation is prognostically adverse. In this setting, mon mesenchymal tumor, where they tend to arise in the 13 Applications of Diagnostic Immunohistochemistry 3 4 3 (A) (B) (C) (D) (E) (F) FIGURE 13.7 Burkitt lymphoma is characterized by a monomorphous infiltrate of intermediate-sized cells with a “starry sky” appearance due to admixed tingible-body macrophages (A). The Ki-67 proliferation index approaches 100% (B) in these tumors, and there is diffuse, strong c-Myc expression, in keeping with MYC activation (C). In contrast, diffuse large B-cell lymphoma demonstrates greater variation in cell size/shape and nuclear contours (D), more variable Ki-67 proliferation indices, in this case 40% to 50% (E), and only patchy staining for c-Myc (F). 3 4 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 13.8 This patient was 4 months status post kidney transplant and presented with dysphagia. A biopsy demonstrates sheets of large cells (A), which express CD20 (B) but not CD3 (C). In situ hybridization for EBV-encoded RNA (EBER) is strongly positive (D). This monomorphic PTLD demonstrates diffuse large B-cell lymphoma histology. Of note, this EBV-negative recipient received a kidney from an EBV-positive donor. muscularis propria (as opposed to the colon, where they and MiTF (though not HMB-45 and only rarely melan-A). typically involve the muscularis mucosae). Expression The tumor’s granular cytoplasm is attributable to massive of the muscle markers SMA and desmin is the rule numbers of lysosomes, and thus CD68 is also expressed. (Figure 13.9A–B). In the esophagus, in particular around The most common mesenchymal tumor in the stom- the gastroesophageal junction, leiomyomas frequently ach is GIST. Gastrointestinal stromal tumors may demon- contain large numbers of KIT-positive non-neoplastic strate spindle cell (70% overall), epithelioid (20% overall; mast cells and interstitial cells of Cajal (the latter are also relatively overrepresented in the stomach), or mixed spin- DOG1-positive), which occasionally leads to a misdiag- dle cell/epithelioid cytomorphology (10% overall). KIT is nosis of GIST (Figure 13.9C). Attention to the distinctive expressed by over 90% of GISTs and is nearly always posi- cytomorphology of leiomyoma should guard against this tive in those with spindle cell morphology (Figure 13.10). diagnostic error, and, of note, desmin is only exception- Tumors that stain weakly for KIT, or are KIT-negative, ally expressed by GIST. usually have PDGFRA mutations and tend to have epithe- The esophagus is also the most common site in the lioid cytomorphology (Figure 13.11). Up to half of KIT- tubal GI tract for granular cell tumors. They express S100 negative GISTs express the calcium-activated chloride 13 Applications of Diagnostic Immunohistochemistry 3 4 5 TABLE 13.6 Immunophenotype of GI Mesenchymal Tumors Tumor type Most Common Location(s) KIT DOG1 CD34 SMA Desmin S100 Other Useful Markers Relatively Common GIST (total) + (92%) + (95%) + (85%) Occ. (20%) Rare (5%) Rare (<1%) GIST, spindle cell type Stomach > small intestine > colon + (98%) + (97%) + (93%) Occ. (20%) Rare (2%) Rare (<1%) > other GIST, epithelioid type Stomach > other sites + (86%) + (92%) + (70%) Occ. (20%) Rare (10%) Rare (<1%) SDHB (deficient in a subset) Leiomyoma Esophagus (muscularis propria) > − − − + + − colon (muscularis mucosae) Mucosal perineurioma Colon (left) − − Occ. − − − EMA+ (typically weak); claudin-1 (50%) Mucosal Schwann cell Colon (left) − − − − − + hamartoma Less Common KIT DOG1 CD34 SMA Desmin S100 Granular cell tumor Esophagus − − − − − + CD68+; MiTF+ Schwannoma Stomach (corpus) > colon − − Rare (10%) − − + GFAP (var.) Plexiform fibromyxoma Stomach (antrum) − − − + Var. (40%) − Glomus tumor Stomach (antrum) − − Occ. (20%) + − − Inflammatory fibroid polyp Small intestine > stomach − − + (85%) Occ. (20%) Rare (5%) − PDGFRA+ (antrum) Kaposi sarcoma Upper > lower GI tract Var. − + − − − HHV8+; CD31+; ERG+ Desmoid-type fibromatosis Mesentery Var. − Rare Var. − − Nuclear β-catenin (70%) Rare KIT DOG1 CD34 SMA Desmin S100 Gastroblastoma Stomach Occ. (20%)* − − − − − Keratins+ (epithelial component); CD10+ (stromal component) Synovial sarcoma Stomach − 10% − Occ. (20%) − Occ. (30%) Keratins/EMA+; TLE1+; t(X;18) FISH Follicular dendritic cell Stomach, colon − − − − − Occ. CD35+; CD21+; CD23 var.; EMA var. sarcoma Clear cell sarcoma-like Small intestine > stomach/colon − − − − − + SOX10+; other melanocytic markers-; tumor of the GI tract synaptophysin (50%); EWSR1 FISH PEComa Colon Rare Rare − Var. Var. Occ. (20%, HMB-45+ more often than melan-A/ focal) tyrosinase/ MiTF/TFE3 Inflammatory Colon, small intestine − − Rare + (90%) + (60%) − ALK (50%); MDM2 Occ.; keratins myofibroblastic tumor (30%, focal) Leiomyosarcoma Colon > small intestine/ − − Occ. + + (75%) − Keratins (30%–40%) esophagus Abbreviation: GIST, gastrointestinal stromal tumor. Key: * in the epithelial component; Rare, ≤10%; Occ., >10% and <30%; Var. ≥30% and <60%. 3 4 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 13.9 Leiomyomas are characterized by fascicles of brightly eosinophilic spindle cells with sharp cell borders (A), and consistently express smooth muscle markers including desmin (B). Tumors around the gastroesophageal junction typically contain large numbers of KIT-staining mast cells and interstitial cells of Cajal, which occasionally leads to an incorrect interpretation of gastrointestinal stromal tumor (C). channel DOG1 (also known as ANO1). This marker is multinodular or plexiform growth pattern, which dem- highly specific for GIST among spindle cells tumors, with onstrate functional deficiency of the Krebs cycle enzyme infrequent and rare positivity reported in synovial sar- succinate dehydrogenase (SDH) (Figure 13.12). SDH- coma and leiomyosarcoma, respectively. CD34 is also fre- deficient GISTs occur in four main clinical settings: quently positive in GIST, especially in spindle cell tumors, • 85% of pediatric GISTs although it is also expressed by vascular and some fibro- • Carney triad (ie, paraganglioma, pulmonary chon- blastic tumors. droma, GIST) Eighty percent of GISTs possess KIT-activating muta- • Carney–Stratakis syndrome (ie, paraganglioma– tions, and another 5% demonstrate PDGFRA-activating GIST dyad) mutations. The remaining 15% are referred to as “wild- • 7.5% of nonsyndromic adult gastric GISTs type.” These wild-type tumors show strong KIT expres- sion immunophenotypically. Among the wild-type tumors Recognition is important because these tumors frequently is a group characterized by female predominance, gastric metastasize (irrespective of traditional risk assessment based location, epithelioid cytomorphology, and a distinctive on location, tumor size, and mitotic rate), do not respond 13 Applications of Diagnostic Immunohistochemistry 3 47 (A) (B) FIGURE 13.10 Classic spindle cell GISTs are disposed as fascicles of palely eosinophilic cells with indistinct cell borders; gastric examples often exhibit perinuclear vacuoles (A). KIT is nearly always expressed by spindle cell GISTs (B). to imatinib, and may have syndromic associations. SDH is expressed. Other gastric mesenchymal tumors in the dif- composed of four protein subunits (SDHA, SDHB, SDHC, ferential diagnosis with GIST include plexiform fibromyx- SDHD). Carney–Stratakis syndrome is autosomal domi- oma and glomus tumor (see also Chapter 5). Plexiform nant, due to an SDH subunit germline mutation; in the other fibromyxoma demonstrates myofibroblastic differentia- three settings, the basis of SDH-deficiency has been linked to tion, and, as such, expresses SMA, while desmin is vari- SDHC promoter methylation. While SDH is normally ubiq- ably expressed. Glomus tumors demonstrate strong SMA uitously expressed, loss of SDHB expression by IHC can be expression, useful in securing the diagnosis. Both of these used to identify SDH-deficient tumors (Figure 13.12C). tumors are KIT negative, as are schwannomas. In the stomach, the closest histologic mimic of GIST Inflammatory fibroid polyps are likely to be encoun- is schwannoma. As elsewhere, schwannomas demonstrate tered in the small intestine as well as the gastric antrum. diffuse, strong S100 expression, while GFAP is variably Investigators have recently identified PDGFRA-activating (A) (B) FIGURE 13.11 Five percent of GISTs possess PDGFRA activating mutations. These tumors tend to be epithelioid (A), and are often KIT-negative or weakly positive (note KIT staining in mast cells) (B). 3 4 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 13.12 SDH-deficient GISTs predominate in the stomach and exhibit a distinctive plexiform/multinodular growth pattern (A) and epithelioid morphology (B). SDH-deficiency is confirmed with an SDHB immunostain (note intact staining in endothelium) (C). mutations in this tumor, supporting its neoplastic nature. differentiation and, as such, demonstrate variable SMA Lesional cells are CD34-positive in 85% of cases, while positivity. Seventy percent of desmoid tumors show SMA is occasionally expressed and desmin is rarely β-catenin nuclear accumulation, as a consequence of either expressed. If available, PDGFRA IHC is positive, though it CTNNB1 or APC mutations (Figure 13.14). Similar to is nonspecific (also positive in GISTs, especially PDGFRA- Kaposi sarcoma, variable KIT staining has been described mutant tumors, and some sarcomas and carcinomas). in desmoid-type fibromatosis. Kaposi sarcoma, when involving the tubal gut, pre- In the colon, mucosal perineurioma and mucosal dominates in the small and large intestine. These tumors Schwann cell hamartoma are small, benign spindle cell express HHV8, as well as vascular endothelial markers lesions of essentially no clinical significance. Their rec- including CD31, CD34, and ERG. Variable KIT staining ognition is important in that the diagnostically unaware has been described, and is especially frequent with antigen may mistake them for ganglioneuroma or neurofibroma, retrieval, in which case the lesion is especially apt to be lesions associated with MEN2B and NF1, respectively. mistaken for GIST (Figure 13.13). Mucosal perineuriomas express EMA (albeit usually quite Desmoid fibromatosis may arise intra-abdominally (ie, weakly) (Figure 13.15) and claudin-1 (in 50%); CD34 is mesentery or pelvis), within the abdominal wall, or extra- occasionally expressed. Mucosal Schwann cell hamarto- abdominally (in somatic soft tissues at any site). Tumors mas strongly

and diffusely express S100, in keeping with may arise sporadically (85% of which show CTNNB1 the presence of schwannian differentiation (Figure 13.16). activating mutations) or in association with familial ade- There are several other rare mesenchymal tumors that nomatous polyposis and related syndromes (due to APC may enter into the morphologic and immunophenotypic mutations). The tumors show fibroblastic/myofibroblastic differential diagnosis in the GI tract. Gastroblastoma is 13 Applications of Diagnostic Immunohistochemistry 3 4 9 (A) (B) (C) (D) (E) (F) FIGURE 13.13 This bland spindle cell lesion in the duodenum obscures the muscularis mucosae and infiltrates the lamina propria; note also the extravasated erythrocytes (A). Based on the presence of KIT staining, this lesion was originally interpreted as a GIST (B), but DOG1 staining was negative (C). CD31 staining confirms the endothelial nature of this lesion (D), and punctate nuclear HHV8 staining supports the diagnosis of KS (E). A week later, a wedge resection from the lung demonstrated similar lesions (F). Based on the diagnosis of KS, the patient was tested and found to have HIV. 3 5 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 13.14 Desmoid tumors are bland spindle cell lesions with collagenous stroma (A); β-catenin nuclear accumulation is detected in 70% of desmoids, distinguishing this tumor from histologic mimics (B). a recently described biphasic gastric tumor composed of Follicular dendritic cell sarcoma presents as a mono- epithelial and stromal elements. The epithelial component morphous proliferation of plump spindle cells with mod- expresses keratins but not EMA, while the spindle cell erate amounts of eosinophilic cytoplasm. Tumor cells component is CD10-positive. KIT and DOG1-positivity demonstrate storiform architecture and are arranged have been described, representing a diagnostic pitfall. syncytially. Frequent tumor-infiltrating lymphocytes are Synovial sarcoma is gastroblastoma’s closest histologic a typical feature. Tumors express markers of follicular mimic, though examples in the stomach are quite rare and dendritic cells including CD35 and CD21, while CD23 is typically monophasic. In addition to patchy EMA and ker- variably expressed. atin expression, synovial sarcomas are characterized by Clear cell sarcoma-like tumor of the GI tract has diffuse, strong TLE1 expression and SS18 rearrangement recently been separated from conventional clear cell sar- by fluorescence in situ hybridization (FISH). coma. Compared to clear cell sarcoma of tendons and (A) (B) FIGURE 13.15 The most consistent marker of mucosal perineurioma (A) is EMA, though expression is often less intense than this (B). 13 Applications of Diagnostic Immunohistochemistry 3 51 (A) (B) FIGURE 13.16 Mucosal Schwann cell hamartomas (A) stain strongly with S100, supporting the schwannian nature of this lesion (B). aponeuroses, the GI variant typically exhibits sheet-like classified as leiomyosarcoma in the tubal gut instead rep- rather than nested architecture, contains osteoclast-like resent GIST. Leiomyosarcomas express SMA and often rather than wreath-like giant cells, and, beyond S100 and express desmin. Keratin and EMA expression are seen in SOX10, it does not express more specific melanocytic 30% to 40%, which represents a diagnostic pitfall. markers (ie, melan-A, HMB-45, MiTF, tyrosinase); both tumor types contain EWSR1 rearrangements. Compared KIT Immunohistochemistry to the much more common metastatic melanoma, clear cell sarcoma exhibits relative monomorphism and lacks Although KIT expression is often considered synonymous macronucleoli. with the diagnosis of GIST, it is expressed by several nor- Perivascular epithelioid cell tumor (PEComa) (see also mal cell types, including mast cells, interstitial cells of Chapter 5) typically presents as a nested proliferation of Cajal, myeloid blasts, germ cells, and melanocytes, as well epithelioid cells with abundant granular eosinophilic or as several other tumors, especially adenoid cystic carci- clear cytoplasm. It demonstrates myomelanocytic differ- noma, myeloid leukemia, seminoma, and some melanomas entiation, and thus is characteristically positive for HMB- (25%–35% of metastases; more often in primary tumors) 45, SMA, and calponin. S100 is less likely to be positive (Figure 13.18; see also Chapter 5). The range of KIT expres- than the more specific melanocytic markers. About 10% sion is narrow in soft tissue tumors, and it is not expressed contain a TFE3 gene fusion, resulting in strong TFE3 by smooth muscle tumors. The list of occasionally KIT- expression. These tumors also occasionally mark with expressing soft tissue tumors includes extraskeletal myxoid KIT, which may lead to diagnostic confusion, but they do chondrosarcoma, Ewing sarcoma/primitive peripheral neu- not mark with DOG1. roectodermal tumors, melanotic schwannoma, low-grade Inflammatory myofibroblastic tumor is typically com- fibromyxoid sarcoma, perineurioma, and angiosarcoma. posed of long fascicles of spindle cells with a prominent KIT is extremely sensitive to heat-induced antigen lymphoplasmacytic inflammatory infiltrate. The most retrieval, and over-retrieval can either decrease expected characteristic immunohistochemical feature is expres- positive staining or, more commonly, result in high-back- sion of ALK, corresponding to ALK gene rearrangement, ground staining leading to nonspecific positivity. Rates though this is seen in only 50% of tumors. Otherwise, of KIT-positivity in desmoid fibromatosis have ranged tumors usually express SMA (90%) and often express from 0 to 100%, due to variations in IHC experimental desmin (60%). MDM2 is occasionally expressed, which conditions. Lucas and colleagues, comparing the perfor- may lead to confusion with dedifferentiated liposar- mance of two commercially available antibodies at three coma, though the latter is associated with greater cyto- separate dilutions with and without antigen retrieval, logic atypia. Leiomyosarcoma (Figure 13.17) is distinctly were able to recapitulate this broad range of positivity. uncommon in the GI tract, and most tumors historically While at low titers, both antibodies marked the majority 3 5 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 13.17 Leiomyosarcomas are typically overtly malignant fascicular spindle cell tumors with eosinophilic cytoplasm and brisk mitotic activity (A); the presence of smooth muscle differentiation is supported by detection of SMA (B) and desmin expression (C). Keratin is expressed by 30% to 40% of leiomyosarcomas, which occasionally leads to an incorrect diagnosis of sarcomatoid carcinoma (D). of desmoids (with accompanying nonspecific stromal ORGAN- AND SITUATION-SPECIFIC staining), at higher titers most tumors were negative. The APPLICATIONS OF IMMUNOHISTOCHEMISTRY best results were achieved with the rabbit polyclonal anti- IN GI TRACT NEOPLASIA body A4502 without antigen retrieval. Similarly, Parfitt and colleagues, examining GI Kaposi sarcomas, recently p53 and Barrett’s-Associated Dysplasia and reported KIT positivity in 4 of 12 (33%) tumors without Carcinoma and 10 of 12 (83%) with antigen retrieval. Like all immu- nostains deployed in the clinical laboratory, KIT should Esophageal adenocarcinoma arises from Barrett’s esopha- be optimized to achieve a high “signal-to-noise ratio,” gus (BE) through a metaplasiaÆdysplasiaÆcarcinoma without excessive background staining (Figure 13.19), sequence (see also Chapter 6). Although the risk of devel- and antigen retrieval is not required as a matter of course. oping adenocarcinoma is only on the order of 0.25% per KIT expression, though characteristic, is not diagnostic year, patients are typically entered into an endoscopic of GIST in spindle cell tumors of the GI tract. As dis- surveillance program. The risk of progression and the cussed above, DOG1 ICH may be complementary in intensity of therapeutic intervention increases signifi- challenging cases. cantly given the detection of dysplasia. BE patients with 13 Applications of Diagnostic Immunohistochemistry 3 5 3 (A) (B) (C) (D) (E) (F) FIGURE 13.18 This ileal spindle cell tumor with perivascular accentuation (A) was originally interpreted as a GIST based on diffuse, strong KIT expression (B). As the morphology was unusual for a spindle cell GIST, additional stains were ordered including S100 (C), which at high power (D) demonstrated focal convincing staining. This result prompted staining for other melanoma markers including HMB-45 (E) and MiTF (F). The patient was subsequently diagnosed with a right forearm melanoma. KIT is not uncommonly expressed by melanoma, presenting a diagnostic pitfall. 3 5 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 13.19 These four images were taken from a desmoid tumor, which should only rarely, if ever, express KIT. A primary antibody dilution of 1:50 without antigen retrieval results in nonspecific background staining without specific staining of mast cells (A). At 1:50 with antigen retrieval, the staining is even stronger, and mast cell staining is now identified (B). At 1:300 without antigen retrieval there is absolutely no staining (C). Staining is optimized at 1:300 with antigen retrieval, which results in specific mast cell staining and a clean background (D). no dysplasia undergo surveillance at intervals of every 3 p53 IHC is among the most well-vetted biomarkers to 5 years. With low-grade dysplasia (LGD), endoscopic used in BE dysplasia assessment, and it has the advantage follow-up is at 6 to 12 month intervals. In patients with of widespread availability. Historically, the use of p53 in high-grade dysplasia (HGD), eradication (eg, radiofre- this context was controversial due largely to lack of uni- quency ablation +/− endoscopic mucosal resection) is typi- formity as to what constituted an abnormal result. More cally recommended. Thus, the prognosis and management recently, investigators have focused on patterns of p53 of BE patients clearly hinges on dysplasia assessment. expression correlating with classes of TP53 abnormali- Unfortunately, however, dysplasia assessment is ties. Most inactivating mutations lead to a p53 confor- fraught with difficulty. Because of this, there is ongoing mational change mitigating protein degradation, leading research to discover and validate objective biomarkers that to high-level nuclear accumulation of a nonfunctioning might increase the accuracy and reproducibility of BE dys- protein (missense-mutation pattern) (Figure 13.20A–B). plasia assessment. Histochemical, immunohistochemical, Less often, destabilizing mutations or gene deletion leads and molecular markers are all being actively investigated. to the complete absence of p53 expression (null pattern) 13 Applications of Diagnostic Immunohistochemistry 3 5 5 (A) (B) (C) (D) (E) (F) FIGURE 13.20 In each of these cases a differential of indefinite for dysplasia (IND) versus low-grade dysplasia (LGD) was contemplated. Case 1: This patient was rebiopsied after a past diagnosis of IND (A); missense-mutation-pattern p53 staining supports an interpretation of LGD (B). Case 2: In this instance, atypia was largely confined to the crypt compartment (C); null-pattern staining again supports an interpretation of LGD (D). Note the single missense-mutation- pattern staining crypt at the lower right. Case 3: This patient carried a prior diagnosis of LGD (E); wild-type pattern staining neither confirms nor refutes a diagnosis of LGD (F). This biopsy was ultimately considered IND. 3 5 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 13.7 Patterns of p53 Expression Pattern Description Biologic Significance Diagnostic Significance Missense-mutation Clonal foci of diffuse, strong High-level accumulation Supports a diagnosis of dysplasia pattern staining obscuring nuclear detail of inactive p53 due to conformational change that prolongs half-life Null pattern Clonal foci of complete absence of Complete absence of expression Supports a diagnosis of dysplasia; in the nuclear staining in a background due to truncating mutations or ProBar study null pattern was associated of wild-type pattern staining large deletions with greater risk of neoplastic progression than missense-mutation pattern Wild-type pattern Weak to moderately intense Physiologic p53 accumulation Does not support, though does not staining, which may be necessarily refute, a diagnosis of punctuated by scattered darkly dysplasia staining nuclei (Figure 13.20C–D). Historically, this pattern was likely to well as application of nonstandardized p53 IHC assess- be misinterpreted as negative. Finally, p53 has a range of ment. The exact frequency of p53 abnormalities remains physiologic expression, with increased expression in the an open question, though it is suspected that they are quite setting of cellular stresses (eg, inflammation, DNA dam- frequent. Thus, p53 IHC is not useful in distinguishing age). This wild-type p53 expression is typically of faint LGD from HGD. The DO-7 mouse monoclonal antibody to moderate intensity, occasionally punctuated by more for p53, among a host of commercially available antibod- darkly staining nuclei (Figure 13.20E–F). It is occasionally ies, has been shown to correlate best with TP53 mutation misinterpreted as positive, though it lacks the uniformity status. of strong nuclear staining and the abrupt/clonal topogra- In a recent prospective case-control study of BE phy of the missense-mutation pattern. These patterns of patients in the Netherlands, p53 overexpression was asso- expression are summarized in Table 13.7. ciated with an adjusted relative risk of 5.6 for progression p53 IHC may be useful in BE cases with atypia in to HGD or adenocarcinoma. Loss of p53 expression (ie, which one is contemplating the differential diagnosis null pattern) was associated with an even higher relative of indefinite for dysplasia versus LGD although its util- risk of 14.0. It is

conceivable that in the future, in addi- ity in this setting remains somewhat controversial and tion to its use in assessing difficult biopsies as discussed is not universally accepted. We find it helpful in difficult in the preceding paragraphs, more routine application of biopsies, including those with active inflammation, those p53 IHC for risk stratification may be incorporated in BE with atypia confined to the crypts, and those in which surveillance programs. Finally, although there is much less the surface is not well-visualized. In this setting, detect- data, we occasionally use p53 IHC in the setting of other ing a missense-mutation or null pattern supports a diag- inflammation-associated atypias in the tubal gut (ie, those nosis of LGD (Algorithm 13.3). In published studies, rates encountered in chronic gastritis and inflammatory bowel of p53 IHC “positivity” have ranged from 9% to 89%; disease), and interpret non-wild-type patterns of p53 stain- moreover, HGD and BE-associated adenocarcinomas are ing in these cases as evidence of neoplasia (Figure 13.21). usually positive. The wide range of p53 abnormalities in As noted previously, however, this practice is somewhat LGD reflects various “thresholds” for LGD diagnosis as controversial and not universally accepted. Epithelial Atypia: Indefinite for dysplasia vs. Low-grade dysplasia Perform p53 IHC Missense-mutation pattern or Null pattern Wild-type pattern Interpretation: Interpretation: Low-grade dysplasia Indefinite for dysplasia ALGORITHM 13.3 Use of p53 Immunohistochemistry to Adjudicate Epithelial Atypia in BE. 13 Applications of Diagnostic Immunohistochemistry 3 57 (A) (B) (C) (D) FIGURE 13.21 Although upper endoscopy demonstrated a linitis-plastica appearance, this biopsy fragment from the corpus appears relatively unremarkable (A). Missense-mutation-pattern p53 staining highlights very subtle involvement by diffuse-type gastric cancer (B). This patient underwent a colectomy for an inflammatory bowel disease-associated adenocarcinoma; p53 staining of the section of distal margin reveals a large field of missense-mutation-pattern staining with rare spared crypts (*) (C). These non-neoplastic crypts (*) are flanked by flat LGD (D). Poorly Differentiated Esophageal Carcinomas factor r eceptor 2 (HER2; see subsequent sections) and anti-VEGF therapy. Also of note, in the seventh edition of In the West, most esophageal cancers are BE-associated the AJCC Cancer Staging Manual, esophageal squamous adenocarcinomas. Poorly differentiated, solid adenocarci- cell carcinoma and adenocarcinoma have separate tumor, nomas may be difficult to distinguish from squamous cell node, metastasis (TNM) stage groupings. carcinomas, especially in small biopsies. Historically, the NECs represent up to 4% of carcinomas in the esoph- distinction of adenocarcinoma from squamous cell carci- agus; these are evenly split between those arising de novo noma in the esophagus had no specific bearing on therapy, and those arising in association with a non-neuroendo- as clinical trials of locally advanced and metastatic tumors crine carcinoma. The treatment paradigm for advanced enrolled patients regardless of histologic type. The distinc- NECs at this site is similar to that for pulmonary small tion has become more significant in the era of directed cell carcinoma. biologic therapy, as only patients with adenocarcinoma Several immunohistochemical markers are useful are potential candidates for anti-human epidermal growth in this situation. Markers of squamous differentiation 3 5 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 13.22 Squamous cell carcinoma is often recognized based on the presence of keratin formation and intercellular bridges (A). Curiously, up to 20% of esophageal (and anal) squamous cell carcinomas demonstrate CDX2-positivity (B); p63 is often coexpressed (C). MUC5AC is the preferred “adenocarcinoma marker” at this anatomic location. include p63 and CK5/6. Both are highly sensitive, though of adenocarcinomas. MOC-31, a monoclonal antibody to not entirely specific, as CK5/6 is noted to be expressed by EPCAM, is sometimes mistakenly interpreted as an ade- approximately 20% of esophageal adenocarcinomas. CK7 nocarcinoma marker in this diagnostic context. Although and CDX2 are often applied as adenocarcinoma markers MOC-31 reliably marks adenocarcinoma, it is expressed at this anatomic site as well. As a note of caution, these by 70% of esophageal squamous cell carcinomas (Figure are expressed by esophageal squamous cell carcinomas in 13.23) and most (60%) NECs. Expression is of greater about 30% and 20% of cases, respectively (Figure 13.22). extent and intensity in poorly differentiated squamous cell Mucin histochemistry may be helpful, though mucin may carcinomas. be very focal to absent in poorly differentiated tumors. As at other sites, IHC for the general neuroendo- MUC5AC appears to be the most specific adenocarci- crine markers chromogranin A and synaptophysin can be noma marker in this setting, though it is only expressed requested when NEC is a consideration. Scattered positive by two-thirds of cases. The embryonic stem cell transcrip- cells should not be overinterpreted as representing a neu- tion factor SOX2 can be used as a marker of squamous roendocrine component. Instead, areas of diffuse, strong cell carcinoma, though it is also expressed by the vast staining should be sought. TTF-1 is expressed by 60% to majority of NECs (greater than 90%) and up to a third 70% of esophageal small cell carcinomas. 13 Applications of Diagnostic Immunohistochemistry 3 5 9 (A) (B) FIGURE 13.23 This keratinizing esophageal squamous cell carcinoma (A) demonstrates strong MOC-31 positivity (B), which, contrary to popular belief, does not support a diagnosis of adenocarcinoma. Overall, 40% of squamous cell carcinomas are MOC-31-positive; the rate is even higher in the esophagus. TABLE 13.8 Immunophenotype of Esophageal to gland-forming tumors, which are more likely to spread Adenocarcinoma Versus Squamous Cell Carcinoma hematogenously to the liver and lung, dyscohesive ones have a tendency to spread transperitoneally to involve Adenocarcinoma Squamous Cell Carcinoma the tubal gut, peritoneal surfaces, and ovaries. Invasive (%, n) (%, n) lobular carcinoma symptomatically involves the GI tract p63 8% (7/92) 99% (66/67) in 0.5% of cases, while in autopsy series metastases to CK5/6 21% (19/92) 97% (65/67) the tubal gut can be found in around half of patients. CDX2 63% (58/92) 19% (13/67) Any segment of the tubal gut may be involved, but there CK7 91% (62/68) 34% (14/41) is a special predilection for the stomach, followed by the MUC5AC 64% (44/69) 2% (1/41) SOX2 29% (27/92) 84% (56/57) large intestine. Clinically evident GI metastases present, on average, 7 to 10 years after the initial presentation Source: Long KB, Hornick JL. SOX2 is highly expressed in squamous cell in the breast. Metastasis to the GI tract is occasionally carcinomas of the gastrointestinal tract. Hum Pathol. 2009;40(12):1768–73; the initial clinical presentation of breast cancer, though, DiMaio MA, Kwok S, Montgomery KD, Lowe AW, Pai RK. Immunohistochemical panel for distinguishing esophageal adenocarcinoma from squamous cell car- and metastases have been reported as far as 30 years out. cinoma: a combination of p63, cytokeratin 5/6, MUC5AC, and anterior gradient Endoscopically, metastatic lobular breast cancer often homolog 2 allows optimal subtyping. Hum Pathol. 2012;43(11):1799–1807. results in a linitis-plastica-like appearance, identical to that produced by diffuse-type gastric cancer; and meta- static lobular breast cancer and diffuse-type gastric cancer When facing a poorly differentiated carcinoma in the are not readily distinguished on H&E evaluation. Thus, it esophagus, a reasonable immunopanel includes p63 and is mandatory to consider a breast metastasis when faced CDX2 (substituting MUC5AC, if available); additional with a diffuse-type anywhere in the tubal gut, but espe- immunostains, including general neuroendocrine mark- cially in the stomach (Figure 13.24A). Background find- ers, can be added as needed. A comparison of the immun- ings such as H. pylori gastritis, intestinal metaplasia, or ophenotype of esophageal adenocarcinoma and squamous epithelial dysplasia in the stomach support a diagnosis of cell carcinoma is presented in Table 13.8. gastric adenocarcinoma. There are several useful immunohistochemical Poorly Cohesive Gastric Cancer Versus markers applicable to this differential diagnosis (Figure 13.24B–C). An initial primary immunopanel often Metastatic Lobular Breast Cancer includes CDX2 and ER. CDX2 is expressed by most gas- Gastric cancer and breast cancer each may present as tric cancers, although typically in fewer cells and of less gland forming or dyscohesive histologic types. Compared intensity than in colon cancer, while it is not expressed 3 6 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) FIGURE 13.24 This 53-year-old woman presenting with nausea, vomiting, and fatigue was found to have a hemoglobin of 5.3. Gastric biopsy demonstrates involvement by a poorly cohesive carcinoma (A). The tumor was found to express ER (B) and not CDX2, consistent with spread from breast. The patient had a negative mammogram, and there was concern over whether or not this represented a breast primary. The tumor was subsequently shown to express GATA3, and a fat- saturated T1-weighted MRI revealed extensive nonmass enhancement of the left breast (C, *) measuring 6 cm and involving the nipple. by lobular breast cancer. ER is usually positive in lobular of the latter). Miettinen and colleagues reported GATA3 breast cancer, and contemporary clones including 1D5, positivity in 5% of 133 gastric cancers, with diffuse-type 6F11, and SP1 are almost never positive in gastric cancer. tumors uniformly negative. A comparison of the immu- In CDX2/ER “double-negative” cases, additional markers nophenotype of poorly cohesive gastric cancer and meta- can be applied including the gastric cancer markers Hep static lobular breast cancer is presented in Table 13.9. Par 1, MUC2, MUC5AC, and CK20, and the breast can- cer markers gross cystic disease fluid protein-15 (GCDFP- HER2 Assessment in Gastroesophageal 15) and mammaglobin. Recently, GATA3 has emerged as a very useful breast cancer marker, and one might consider Adenocarcinoma adding it to the initial CDX2/ER panel or substituting it Numerous factors culminated in an international, phase for ER. GATA3 is less likely to be positive in ER-negative 3, open-label, randomized controlled trial comparing than in ER-positive breast cancers, though in our experi- trastuzumab plus standard chemotherapy (fluoropy- ence it is expressed by 70% to 80% of the former (and all rimidine + cisplatin) to chemotherapy alone in advanced 13 Applications of Diagnostic Immunohistochemistry 3 61 TABLE 13.9 Immunophenotype of Poorly Cohesive Gastric Cancer Versus Metastatic Lobular Breast Cancer Poorly Cohesive Metastatic Gastric Cancer (%, n) Lobular Breast Cancer (%, n) CDX2 78% (42/54) 0% (0/51) ER 0% (0/58) 76% (42/55) Hep Par1 83% (25/30) 0% (0/21) GCDFP-15 0% (0/28) 76% (26/34) MUC1 20% (10/51) 100% (27/27) MUC2 51% (40/79) 13% (8/60) MUC5AC 54% (43/79) 5% (3/60) MUC6 35% (17/49) 10% (4/39) CK7 66% (38/58) 100% (21/21) CK20 53% (31/58) 5% (1/20) E-cadherin (intact) 57% (17/30) 29% (6/21) GATA3 0% of diffuse-type tumors 71% (867/1229)b 5% overall (6/133) including intestinal-type tumorsa Source: Chu PG, Weiss LM. Immunohistochemical characterization of signet-ring cell carcinomas of the stomach, breast, and colon. Am J Clin Pathol. 2004;121(6):884–892; O’Connell FP, Wang HH, Odze RD. Utility of immunohistochemistry in distinguishing primary adenocarcino- mas from metastatic breast carcinomas in the gastrointestinal tract. Arch Pathol Lab Med. 2005;129(3):338–347; Nguyen MD, Plasil B, Wen P, Frankel WL. Mucin profiles in signet-ring cell carcinoma. Arch Pathol Lab Med. 2006;130(6):799–804; aMiettinen M, McCue PA, Sarlomo-Rikala M, et al. GATA3: a multispecific but potentially useful marker in surgical pathology: a systematic analysis of 2500 epithelial and nonepithelial tumors. Am J Surg Pathol. 2014;38(1):13–22; bOrdonez NG. Value of GATA3 immunostaining in tumor diagnosis: a review. Adv Anat Pathol. 2013;20(5):352–360. (ie, locally advanced, metastatic, or recurrent) gastric of the ToGA trial. More frequent HER2 heterogeneity and gastroesophageal junction (GEJ) adenocarcinomas, (areas of positive and negative staining) was observed in known as the Trastuzumab for Gastric Cancer (ToGA) gastroesophageal tumors than had been seen in breast trial. These factors included an observed rate of HER2 tumors (Figure 13.25). Also, several cases with basolat- overexpression in gastric cancer similar to that seen in eral (U-shaped) or lateral membrane staining (rather than breast cancer; the efficacy of trastuzumab (Herceptin®) complete membrane staining) were observed, and some in preclinical models of gastric cancer; and a pau- of these were HER2-amplified by FISH. Because of het- city of effective therapies in advanced gastric cancer. erogeneity, there are different scoring criteria for gastro- For the purpose of the trial, cases were tested by both esophageal biopsies and resections, with any amount of IHC and FISH. Patients with an IHC score of 3+ and/ 2+ or 3+ staining in a biopsy considered equivocal or posi- or with a FISH HER2:CEP17 ratio of >/=2 were consid- tive, respectively (one group has alternatively suggested ered positive. Among 3,807 tumors screened for enroll- the need for at least five cells that stain—key difference ment, HER2-positivity was seen in 20.9% of gastric and 1). Because cases with basolateral and lateral

membrane 33.2% of gastroesophageal junction tumors. Histologic staining may be HER2-amplified, the strict requirement type influenced expression, with positivity in 32.2% of for “complete membrane staining” that applies in breast intestinal, 20.4% of mixed, and only 6.1% of diffuse cancer is relaxed (key difference 2). GI HER2 scoring tumors. The addition of trastuzumab in HER2-positive criteria are summarized in Table 13.10 and illustrated in patients improved median overall survival by 2.7 months Figure 13.26. (13.8 months vs. 11.1 months). This “positive” study has In the ToGA trial, the survival benefit from trastu- resulted in the assessment of HER2 in advanced gastric zumab was greatest in patients with IHC 3+ or IHC 2+/ and gastroesophageal adenocarcinomas becoming the FISH-positive tumors (4.2 months), while patients with standard of care. The National Comprehensive Cancer HER2 amplification in the absence of protein overexpres- Network (NCCN) has further extended this recommen- sion (IHC 0/1+) derived no survival benefit. This latter dation to patients with esophageal adenocarcinoma. In group constituted 22% of all HER2-positive cases. Based the near future, it is anticipated that anti-HER2 therapy on this result, many laboratories have established HER2 in HER2-positive tumors will be explored in clinical tri- IHC as the first-line test, with FISH principally reserved als as a component of neoadjuvant regimens. for HER2 2+ (equivocal) cases. Other laboratories have There are two key differences between HER2 IHC taken the stance that the biology of HER2 IHC-negative/ testing in gastroesophageal cancer and breast cancer. FISH-positive cases is still an open question and either These are based, in part, on observations made in the perform IHC and FISH in parallel or reflex HER2 0 and context of a validation study performed in anticipation 1+ (in addition to 2+) cases to FISH. 3 6 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 13.25 Gastroesophageal adenocarcinomas occasionally demonstrate HER2 heterogeneity, as seen in this example where there are areas of 3+, 2+, and 1+ staining in the same tissue section (A), while breast tumors, if positive (3+), tend to be uniformly so (B). This heterogeneity in GI tumors is the basis of separate scoring criteria in biopsies and resections and repeating HER2 testing on resection specimens from patients who have had negative results on biopsy. At our institution, we perform HER2 testing on all the ToGA trial screened tumors with the Food and Drug advanced esophageal, gastroesophageal junction, and gas- Administration (FDA)-approved Dako HercepTest™ (rab- tric adenocarcinomas, and some perform testing on nonad- bit polyclonal), the 4B5 antibody (rabbit monoclonal) has vanced cases as well. Potential specimens include endoscopic been shown to produce equivalent results. The College of mucosal biopsies, resection specimens, and biopsies of American Pathologists provides proficiency testing for GI metastatic sites. Studies of matched endoscopic mucosal HER2 IHC. biopsies and resection specimens and matched primary and metastatic tumors have reported concordances of 75% to 95%. Given an initially negative result, it may be prudent to MISMATCH REPAIR PROTEIN test more than one specimen, particularly if a biopsy from IMMUNOHISTOCHEMISTRY a metastatic site becomes available, as metastatic tumors have been reported to convert to HER2-positive 10% of the Malignant neoplasms are characterized by genomic insta- time. Similar to breast cancer specimens, it is recommended bility, which takes two main forms (see also Chapter 14). that samples be placed in 10% neutral buffered formalin Colorectal cancers can be divided into those demonstrating within 1 hour and fixed for 6 to 72 hours, with the fixa- chromosomal instability (85%) and those demonstrating tion time documented in the pathology report. Although microsatellite instability (MSI; 15%). MSI occurs due to TABLE 13.10 HER2 Immunohistochemistry Scoring Criteria for Resections and Biopsies Biopsy Resection Score Interpretation Action No reactivity No reactivity or reactivity in 0 Negative Some laboratories will reflex to <10% of tumor cells HER2 FISH Tumor cell cluster with faint/barely perceptible Faint/barely perceptible 1+ Negative Some laboratories will reflex to reactivity (ie, discernible at 400x) reactivity in ≥10% of tumor HER2 FISH cells Tumor cell cluster with weak to moderate Weak to moderate reactivity 2+ Equivocal Reflex to HER2 FISH reactivity (ie, discernible at 100–200x) in ≥10% of tumor cells Tumor cell cluster with strong reactivity Strong reactivity in ≥10% of 3+ Positive Consider anti-HER2 therapy (ie, visible with naked eye or discernible at tumor cells 20–40x) Note: Significant reactivity includes complete, lateral, or basolateral membrane staining; in one study, a tumor cell cluster was defined as five or more cells. Abbreviation: FISH, fluorescence in situ hybridization. 13 Applications of Diagnostic Immunohistochemistry 3 6 3 (A) (B) (C) (D) (E) FIGURE 13.26 In gastroesophageal adenocarcinomas, a score of zero equals no reactivity (A); 1+: faint/barely perceptible reactivity (B); 2+: weak to moderate reactivity (C); 3+: strong reactivity (D). The 2+ biopsy illustrated in “C” was reflexed to FISH, which demonstrates a HER2 (orange) to CEP17 (green) ratio of 3.0 (ie, amplified) (E). Note that in the 1+ to 3+ cases, staining is predominantly lateral or basolateral, generally sparing the apical surface. 3 6 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide deficient DNA mismatch repair (dMMR) function, which due to Lynch syndrome are typically counseled to undergo may arise sporadically (typically due to methylation of the subtotal rather than segmental colectomy due to a sub- MLH1 promoter and seen in up to 15% of colon cancers) stantial (~40%) risk of metachronous tumors. In 80% to or in the setting of germline mutations (ie, Lynch syn- 85% of colon cancer cases, all four proteins are normally drome). The identification of colon cancers with dMMR/ expressed (pattern 1). Because in rare Lynch syndrome MSI-H is desirable to identify Lynch syndrome (2–4% of patients a nonfunctioning protein is still expressed, com- colon cancers) but also because this phenotype is prognos- plementary MSI testing may be considered in patients tically favorable stage for stage and predicts a relative lack with normal IHC but clinical suspicion of Lynch syn- of benefit from 5-FU-based chemotherapy. Either poly- drome. Loss of MLH1 and PMS2 is seen in 15% of cases merase chain reaction (PCR)-based MSI testing or MMR (pattern 2). This is usually due to MLH1 promoter meth- protein IHC may be used as screening tests for the dMMR/ ylation and is less often due to MLH1 germline muta- MSI-H phenotype, with highly concordant results. MMR tion. BRAF V600E and/or MLH1 promoter methylation IHC offers the advantages of widespread availability and testing can be pursued in this setting to distinguish spo- rapid turnaround time, and, when abnormal, it directs the radic from hereditary cases. In tumors with wild-type next steps in the patient’s evaluation and plan for therapy. BRAF and/or without demonstrable MLH1 promoter Although age, clinical history, and histology-based methylation, MLH1 germline mutation testing is then strategies to identify which tumors to screen for dMMR/ undertaken. Until recently, patterns 3 to 5 were consid- MSI-H may be employed, there has been a trend toward ered tantamount to a diagnosis of Lynch syndrome, with more intensive, if not universal, screening. The Centers germline mutation testing directed by the specific result for Disease Control and Prevention (CDC)-sponsored of the IHC. Evaluation of Genomic Applications in Practice and In at least 30% of patients with suspected Lynch syn- Prevention (EGAPP) working group and the Association drome (abnormal MMR IHC and/or MSI-H in whom a for Molecular Pathology (AMP) Mismatch Repair- sporadic tumor due to MLH1 promoter methylation has Defective CRC working group have endorsed universal been excluded), a germline mutation is not detected. This screening. The most recent NCCN Guidelines recom- had been ascribed to the relative insensitivity of germ- mend (a) testing tumors in patients aged 70 and less, as line mutation testing. Several groups have recently shown well as those above 70 who meet Bethesda guidelines or that in up to two-thirds of these cases biallelic inactiva- (b) universal testing. The NCCN guidelines also suggest tion of an MMR gene arises somatically (ie, only within that testing may be helpful in patients with stage II colon the tumor). These cases have been referred to as “Lynch- cancer, in which the result influences the decision as to like” or “pseudo-Lynch” syndrome. Family members of whether to pursue adjuvant chemotherapy. patients with Lynch-like syndrome have a risk of colon The DNA MMR apparatus is one of several DNA cancer intermediate between that seen in families with repair mechanisms. It specifically recognizes mispaired Lynch syndrome and those in which colon cancer appears bases and insertion–deletion loops, which, if unrepaired, to have arisen sporadically, and Lynch-like syndrome is would lead to missense and frameshift mutations, respec- not associated with the typical Lynch-associated extraco- tively. The system functions as two heterodimers, with lonic tumors (eg, carcinomas of the endometrium, ovary, MSH2 pairing with MSH6 to recognize the errors and stomach, and upper urinary tract). MLH1 pairing with PMS2 to direct the repair. Each het- Historically, patients with abnormal MMR IHC and/ erodimer is composed of a dominant (expressed regardless or MSI testing that remained “unexplained” (ie, BRAF of the status of its partner) and dependent (not expressed wild-type, MLH1 promoter methylation negative, germ- in the absence of its partner) protein; MLH1 and MSH2 line DNA MMR mutation negative) were followed as are dominant, while PMS2 and MSH6 are dependent. if they had Lynch syndrome (ie, intensive colonoscopy, Biallelic MLH1 promoter methylation silences MLH1 endometrial sampling, transvaginal ultrasound, urine (and PMS2) expression. In Lynch syndrome, a germline cytology). Because potentially affected family members mutation and a somatic “second hit” silences expression could not be effectively evaluated for Lynch syndrome of one or more proteins in ~95% of cases. There are five due to the lack of an identifiable mutation, they too, main patterns of MMR protein expression seen in tumors: were often placed into intensive surveillance. The iden- tification of Lynch-like syndrome, by testing the tumors 1. All four proteins expressed of patients with unexplained MMR IHC and/or MSI 2. Loss of MLH1/PMS2; intact MSH2/MSH6 results for somatic DNA MMR gene mutations, prom- 3. Loss of MSH2/MSH6; intact MLH1/PMS2 ises to be cost-effective, sparing patients and their fam- 4. Loss of MSH6; intact MLH1/PMS2/MSH2 ily members the expense of Lynch-syndrome-specific 5. Loss of PMS2; intact MLH1/MSH2/MSH6 surveillance. Patterns of MMR protein IHC and their We perform MMR IHC on biopsies (if available) frequency, interpretation, and follow-up are summa- rather than resections because patients with colon cancer rized in Table 13.11. 13 Applications of Diagnostic Immunohistochemistry 3 6 5 TABLE 13.11 Patterns of Mismatch Repair Protein Expression Immunohistochemistry Result Frequency Interpretation Action(s) All four proteins intact 80%–85% Normal MMR function Consider follow-up microsatellite instability Unlikely Lynch syndrome testing to confirm normal result Refer to Cancer Genetics if clinically appropriate MLH1/PMS2 lost 15% Abnormal MMR function BRAF V600E and/or MLH1 promoter MSH2/MSH6 intact Likely sporadic dMMR due to methylation testing MLH1 promoter methylation If BRAF V600E and/or MLH1 promoter Less likely Lynch syndrome due to methylation testing are normal: MLH1 (usually) or PMS2 (rarely) Refer for genetics evaluation mutation MLH1 germline mutation testing (followed by PMS2 if needed) Consider tumor mutation testing if germline mutation not detected MSH2/MSH6 lost 1%– 2% Abnormal MMR function Refer for genetics evaluation MLH1/PMS2 intact Possibly Lynch syndrome due MSH2 germline mutation testing (followed to MSH2 (usually) or EPCAM by EPCAM and MSH6 if needed) deletion or MSH6 mutation Consider tumor mutation testing if germline (rarely) mutation not detected MSH6 lost Up to 0.5% Abnormal MMR function Refer for genetics evaluation MLH1/PMS2/MSH6 intact Possibly Lynch syndrome due to MSH6 germline mutation testing (followed by MSH6 (usually) or MSH2 (rarely) MSH2 if needed) mutation Consider tumor mutation testing if germline mutation not detected PMS2 lost up to 0.5% Abnormal MMR function Refer for genetics evaluation MLH1/MSH2/MSH6 intact Possibly Lynch syndrome due to PMS2 germline mutation testing PMS2 (usually) or MLH1 (rarely) (followed by MLH1 if needed) mutation Consider tumor mutation testing if germline mutation not detected Abbreviations: dMMR, deficient mismatch repair function; MMR, mismatch repair. Investigators have noted several potential pitfalls in MMR proteins may rarely show similar block-like/clonal the interpretation of MMR IHC. A determination of loss loss, corresponding to the unusually late acquisition of the of protein expression can only be made in the setting of second hit to an MMR gene at the carcinoma stage. intact staining in internal control lymphocytes and stroma. MMR protein expression may be difficult to

detect EPCAM Immunohistochemistry (including in internal control tissue) with some fixatives other than formalin (Figure 13.27A). Protein expression Recently, large deletions in the 3’ end of EPCAM have may be heterogeneous, especially in resections as com- been found to account for about 20% of Lynch syndrome pared to biopsies (attributed to delays in fixation or under/ cases in patients with loss of MSH2/MSH6 expression overfixation) (Figure 13.27B–C), and after neoadjuvant without identifiable MSH2 germline mutations. EPCAM chemoradiotherapy (MSH6 is especially prone to this, and lies directly upstream of MSH2, and these large deletions may demonstrate a peculiar nucleolar pattern of expres- lead to MSH2 promoter methylation and transcriptional sion; Figure 13.27D–E). In general, MMR IHC should silencing. There is loss of EPCAM expression in affected only be considered abnormal in the setting of complete tumors, with intact expression in non-neoplastic crypts. absence of one or more proteins. Rarely, one or more pro- The monoclonal antibody BerEp4 recognizes EPCAM and teins demonstrate diffuse, weak staining, which is weaker may be used to identify MSH2/MSH6-deficient tumors than associated internal control tissue (Figure 13.27F–G). due to EPCAM mutation. These cases may cautiously be interpreted as abnormal, likely due to mutations that abrogate protein function and BRAF V600E Mutation-Specifi c diminish, although not entirely silence, protein expres- Immunohistochemistry sion. MSH6 has microsatellites in its coding region, and it is occasionally silenced as a secondary event in dMMR/ A BRAF V600E mutation-specific monoclonal antibody MSI-H tumors (ie, MSH6 loss, which may be block-like/ has recently become commercially available. Several clonal rather than diffuse, may accompany loss of MLH1/ groups have published near-perfect to perfect sensitivity PMS2 or loss of PMS2; Figure 13.27H). Loss of the other and specificity for this antibody in, collectively, several 3 6 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) (E) (F) FIGURE 13.27 (continued) 13 Applications of Diagnostic Immunohistochemistry 3 67 (G) (H) FIGURE 13.27 Absent internal control staining with use of a nonformalin, glyoxal-based fixative; this result is uninterpretable (A). Variation in staining is not unusual in resection specimens (B), including areas of very focal and weak, though intact, staining (C). Reduced staining intensity and peculiar nucleolar localization may be seen after neoadjuvant chemoradiotherapy; MSH6 is preferentially affected (D); MSH6 staining in a matched pretreatment biopsy is clearly intact (E). This tumor demonstrates uniform weak MSH6 staining in tumor (left side), relative to strongly staining internal control (right side) (F), while MSH2 staining intensity is similar in tumor and internal control (G); this tumor was subsequently shown to be MSI-H. Clonal loss of MSH6 as a secondary event in a tumor that was uniformly MLH1/PMS2-deficient (H). thousand colon cancers. Of note, the assay appears most IHC, even small polyps with LGD are often abnormal. likely to achieve these superior test characteristics when Testing adenomatous polyps may be useful in patients with run on Ventana automated immunostainers. In con- family histories compelling for Lynch syndrome in whom trast, a group using manual staining reported a sensitiv- cancer tissue is unavailable, as an abnormal result is fairly ity and specificity of only 71% and 74%, respectively, sensitive and highly specific for Lynch syndrome. However, in a cohort of 52 tumors, and another group, using a routine testing of adenomatous polyps in patients without Leica Bond-Max autostainer found moderate to strong any clinical or family history that is compelling for Lynch staining with the antibody to be 85% sensitive and 68% syndrome is not recommended. specific in a group of 113 colon cancer patients. BRAF Sporadic MSI-H colon cancers due to MLH1 pro- V600E IHC may substitute for BRAF mutation analy- moter methylation are characterized by loss of MLH1/ sis in the evaluation of MLH1-deficient colon cancers, PMS2 expression. These tumors arise from sessile serrated but the assay must be carefully validated, and it may not polyps, which develop cytologic dysplasia before becom- achieve acceptable test characteristics on some testing ing invasive cancer. While sessile serrated polyps with- platforms. Interestingly, papillary thyroid cancers and out cytologic dysplasia demonstrate intact MLH1/PMS2 melanomas may not be susceptible to the same platform- protein expression, loss is demonstrated in most foci of specific variability. superimposed HGD. The frequency of protein loss in ses- sile serrated polyp with LGD is not well-established, but, Mismatch Repair Immunohistochemistry in our experience, it is relatively infrequent (Figure 13.29). in Colon Polyps Patients with Lynch syndrome inherit one defective copy of a DNA MMR gene. The second hit occurs in adenoma- ANAL SQUAMOUS INTRAEPITHELIAL tous polyp tissue sometime after polyp initiation, and has LESIONS/INTRAEPITHELIAL nearly always occurred by the time cancer develops. MMR NEOPLASIA (SIL/AIN) protein deficiency (or MSI-H) may be detected in 60% to 80% of adenomas from patients with Lynch syndrome Anogenital squamous cell carcinoma is etiologically (Figure 13.28). Although larger polyps and those harbor- linked to high-risk human papillomavirus. Neoplastic ing HGD are more likely to demonstrate abnormal MMR progression is driven, in part, by production of the E6 3 6 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 13.28 Loss of MSH2 (A) and MSH6 expression in a small adenoma from a woman with a history of endometrial cancer and sebaceous adenoma. Intact mismatch repair protein expression (B, MSH2) in the same patient in a separate polyp from the same colonoscopy. and E7 oncoproteins, the latter of which inactivates the and/or “spotty.” Block-like staining has been described in 0% tumor suppressor retinoblastoma protein (pRb). p16 is to 21.4% (median 9.5%) of cases of AIN1. Although AIN1 upregulated in the face of pRb inactivation, and p16 IHC is typically associated with low-risk HPV, some of these is therefore widely used as a surrogate marker of high risk p16-positive cases have been shown to harbor high risk HPV. HPV-driven squamous lesions (eg, in the uterine cervix, In addition to distinguishing HSIL from AIN1, p16 oropharynx, and anus) (see also Chapter 12). IHC is also useful in separating HSIL from anal transi- p16 has been shown to be overexpressed in 76% to tional zone (ATZ) mucosa. Although the anal squamoco- 100% (median 86%) of high-grade squamous intraepithe- lumnar junction may be abrupt, stretches of a multilayered lial lesions (HSIL; encompassing AIN2 and AIN3). Positive epithelium, four to nine cells thick, often with a fairly staining is “block-like” (diffuse, strong), rather than weak high nucleus–cytoplasm ratio, and variously composed (A) (B) FIGURE 13.29 Sessile serrated polyp (upper right) with superimposed low-grade cytologic dysplasia (lower left) (A); MLH1 immunostain demonstrates loss of expression in the area of cytologic dysplasia (B). 13 Applications of Diagnostic Immunohistochemistry 3 6 9 (A) (B) FIGURE 13.30 Anal transitional zone mucosa, given the often moderately high nucleus:cytoplasm ratio, is occasionally overinterpreted as HSIL (A). p16-negativity argues against the diagnosis of HGSIL (B). of squamous, transitional, and mucous cells, may be mis- thickness. Ki-67 performs similarly, though not quite taken for HSIL (Figure 13.30A). Two studies have shown as well, as p16, because inflammatory/reactive changes ATZ mucosa to be p16-negative (Figure 13.30B); it is also sometimes stain in a pattern that may be interpreted as CK7-positive, while HSIL rarely is. “positive.” In noninflamed ATZ mucosa, staining is char- Several studies have also evaluated the efficacy of acteristically confined to the basal layer. Ki-67 IHC, generally in concert with p16, to adjudicate We typically perform p16 IHC (sometimes along with anal squamous lesions. Various definitions of “positive” Ki-67) in small biopsies in which we are uncertain on the have been used, including staining of greater than one H&E as to whether HSIL is present. Block-like staining third or greater than one half of the lesional epithelial supports a diagnosis of HSIL (Figure 13.31). (A) (B) FIGURE 13.31 This patient had a history of squamous cell carcinoma in situ of the anus, which had been irradiated. Follow-up biopsy contained extremely scant squamous epithelium (A), which demonstrated block-pattern p16 staining (B, right side of biopsy), supporting the diagnosis of HSIL. 370 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Pagetoid Tumors of the Anal Canal Extramammary Paget disease is a histologically similar lesion, often presenting in the anogenital region. In con- In 1874, the English surgeon and pathologist James Paget trast to mammary Paget disease, it often represents a pri- reported an association between chronic nipple ulceration mary intraepithelial adenocarcinoma, which rarely invades and the subsequent development of breast cancer. It was (see also Chapter 12). Primary anogenital Paget disease later shown that these nipple–areolar changes were neo- plastic, rather than inflammatory, and in fact an under- must be distinguished from secondary involvement of ano- lying in situ or invasive carcinoma is identified in more genital epithelium by an underlying visceral malignancy than 95% of patients with Paget disease of the nipple. (eg, anal duct, colorectal, or genitourinary). The differen- Histologically, Paget disease is characterized by intraepi- tial diagnosis of pagetoid lesions also includes melanoma dermal involvement by cytologically malignant cells, sin- in situ/superficial spreading melanoma and some examples gly and in small groups, which tend to predominate at the of squamous cell carcinoma in situ. base and disperse toward the epithelial surface (so-called In challenging cases, this differential diagnosis may be “upward spread”). This histologic appearance is referred readily solved immunohistochemically (Figure 13.32B–D). to as “pagetoid” (Figure 13.32A). Primary Paget disease of the anus reliably expresses CK7, (A) (B) (C) (D) FIGURE 13.32 Malignant-appearing cells with abundant eosinophilic cytoplasm involve the anal squamous epithelium singly and in small groups (A); the neoplastic population expresses CK20 (B) and CDX2 (C), but not GATA3 (D; note weak expression in the squamous epithelium, however). This immunophenotype suggests pagetoid involvement of the anal canal mucosa by a lower GI tract adenocarcinoma. 13 Applications of Diagnostic Immunohistochemistry 371 TABLE 13.12 Immunophenotype of Neoplasms Involving Perianal Area in Pagetoid Fashion Neoplasm Positive Stains Comments Extramammary Paget disease CK7, GATA3, HER2 (var.), GCDFP-15 (var.), androgen Reportedly rarely may receptor (var.) be CK20+ Colorectal adenocarcinoma involving anal CK20, CDX2 May be CK7+ mucosa/perianal skin in pagetoid fashion Pagetoid squamous cell carcinoma in situ p63, CK5/6, p16 May be CK7+ Melanoma in situ/superficial spreading S100, melan-A/MART-1, HMB-45, MiTF, SOX10, melanoma tyrosinase while HER2, GCDFP-15, and androgen receptor are the perianal area in pagetoid fashion is summarized in variably expressed. Recently, in two small studies pub- Table 13.12. lished in abstract form, GATA3 was shown to be uni- formly expressed by extramammary Paget disease. Rare Basaloid Squamous Cell Carcinoma Versus CK20-positive examples of extramammary Paget disease Basal Cell Carcinoma at the Anal Margin have been published, although it is possible that in these instances an underlying colorectal carcinoma escaped Rarely, we have encountered small biopsies in which the clinical detection. Although mucin histochemistry distin- distinction of basaloid squamous cell carcinoma from guishes extramammary Paget disease from melanocytic basal cell carcinoma (BCC) has proven challenging (see and squamous examples, it will not distinguish it from also Chapter 12). The distinction is clinically significant, secondary involvement by an underlying adenocarci- because invasive anal squamous cell carcinoma is treated noma. Colorectal adenocarcinomas will generally express with radiation and chemotherapy (eg, 5-FU), while BCC CK20 and CDX2 (80%–90%), and aside from expressing is treated with wide local excision. Of note, BCC only CK7 in up to 25% of cases, they would not be expected to rarely arises at this anatomic location but it does occur. express any of the other “extramammary Paget disease- A recent study examined the ability of histologic features specific” markers. Pagetoid squamous cell carcinoma in and a panel of immunohistochemical stains to distinguish situ expresses p63 and CK5/6, and rare examples have these tumors. There was substantial morphologic over- been shown to express CK7, leading to diagnostic con- lap, with the only statistically significant distinguishing fusion with extramammary Paget disease. Melanocytic morphologic features being retraction of nests of tumor lesions express the array of melanocyte markers includ- from the adjacent stroma (BCC) and the presence of atypi- ing S100, melan-A/MART-1, and HMB-45. cal mitotic figures (basaloid squamous cell carcinoma). Our primary immunopanel, given a pagetoid neoplasm Basaloid squamous cell carcinomas expressed p16 and at the anal margin, includes CK7, CK20, CDX2, p63, and SOX2, while BCCs did not; BerEp4 and Bcl-2 were more S100. Additional markers can be added, as needed. Given frequently expressed in BCCs; and p63 and CK5/6 were recent findings, we would consider substituting GATA3 frequently expressed by both tumor types (see

Table 13.13 for CK7. The immunophenotype of neoplasms involving and Figure 13.33). TABLE 13.13 Immunophenotype of Anal Region Basaloid Squamous Cell Carcinoma Versus Basal Cell Carcinoma Basaloid Squamous Cell Carcinoma (%, n) Basal Cell Carcinoma (%, n) p16 93% (14/15) 0% (0/9) SOX2 93% (14/15) 0% (0/9) BerEp4 40% (6/15) 100% (9/9) Bcl-2 33% (5/15) 100% (9/9) p63 87% (13/15) 100% (9/9) CK5/6 80% (12/15) 100% (9/9) Source: Patil DT, Goldblum JR, Billings SD. Clinicopathological analysis of basal cell carcinoma of the anal region and its distinction from basaloid squamous cell carcinoma. Mod Pathol. 2013;26(10):1382–1389. 372 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) (C) (D) FIGURE 13.33 Basaloid squamous cell carcinoma presents as nests of cells with a high nucleus:cytoplasm ratio and brisk mitotic activity (A); diffuse, strong p16 expression, etiologically linked to high-risk human papillomavirus, confirms the morphologic impression (B). Characteristic retraction artifact is inconspicuous in this infiltrative BCC of the perianal skin (C); BerEp4 positivity is typical, though it may also be seen in anogenital squamous cell carcinomas (D). SELECTED REFERENCES Goldstein NS, Bassi D. Cytokeratins 7, 17, and 20 reactivity in pancre- atic and ampulla of vater adenocarcinomas. Percentage of positiv- ity and distribution is affected by the cut-point threshold. Am J Approach to Epithelial Neoplasms Clin Pathol. 2001;115(5):695–702. Adams H, Schmid P, Dirnhofer S, Tzankov A. Cytokeratin expres- Gustmann C, Altmannsberger M, Osborn M, Griesser H, Feller AC. sion in hematological neoplasms: a tissue microarray study Cytokeratin expression and vimentin content in large cell anaplas- on 866 lymphoma and leukemia cases. Path Res Prac. tic lymphomas and other non-Hodgkin’s lymphomas. Am J Pathol. 2008;204(8):569–573. 1991;138(6):1413–1422. Chen ZM, Wang HL. Alteration of cytokeratin 7 and cytokeratin 20 Herrera GA, Turbat-Herrera EA, Lott RL. S-100 protein expression expression profile is uniquely associated with tumorigenesis of pri- by primary and metastatic adenocarcinomas. Am J Clin Pathol. mary adenocarcinoma of the small intestine. Am J Surg Pathol. 1988;89(2):168–176. 2004;28(10):1352–1359. Ji H, Isacson C, Seidman JD, Kurman RJ, Ronnett BM. Cytokeratins Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expres- 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic sion in epithelial neoplasms: a survey of 435 cases. Mod Pathol. mucinous carcinomas in the ovary from primary ovarian mucinous 2000;13(9):962–972. tumors: Dpc4 assists in identifying metastatic pancreatic carcino- Chu PG, Schwarz RE, Lau SK, Yen Y, Weiss LM. Immunohistochemical mas. Int J Gyn Pathol. 2002;21(4):391–400. staining in the diagnosis of pancreatobiliary and ampulla of Vater Laury AR, Perets R, Piao H, et al. A comprehensive analysis of adenocarcinoma: application of CDX2, CK17, MUC1, and MUC2. PAX8 expression in human epithelial tumors. Am J Surg Pathol. Am J Surg Pathol 2005;29(3):359–367. 2011;35(6):816–826. 13 Applications of Diagnostic Immunohistochemistry 373 Logani S, Oliva E, Arnell PM, Amin MB, Young RH. Use of novel van Velthuysen ML, Groen EJ, van der Noort V, van de Pol A, immunohistochemical markers expressed in colonic adenocar- Tesselaar ME, Korse CM. Grading of Neuroendocrine Neoplasms: cinoma to distinguish primary ovarian tumors from metastatic Mitoses and Ki-67 both Essential. Neuroendocrinology. colorectal carcinoma. Mod Pathol. 2005;18(1):19–25. 2014;100(2–3):221–227. Lugli A, Tzankov A, Zlobec I, Terracciano LM. Differential diagnostic and functional role of the multi-marker phenotype CDX2/CK20/ CK7 in colorectal cancer stratified by mismatch repair status. Mod Approach to Hematolymphoid Tumors Pathol. 2008;21(11):1403–1412. Bagdi E, Diss TC, Munson P, Isaacson PG. Mucosal intra-epithelial Magnusson K, de Wit M, Brennan DJ, et al. SATB2 in combination lymphocytes in enteropathy-associated T-cell lymphoma, ulcer- with cytokeratin 20 identifies over 95% of all colorectal carcino- ative jejunitis, and refractory celiac disease constitute a neoplastic mas. Am J Surg Pathol. 2011;35(7):937–948. population. Blood. 1999;94(1):260–264. Nonaka D, Chiriboga L, Rubin BP. Sox10: a pan-schwannian and mela- Chan JK, Chan AC, Cheuk W, Wan SK, Lee WK, Lui YH, et al. Type II nocytic marker. Am J Surg Pathol. 2008;32(9):1291–1298. enteropathy-associated T-cell lymphoma: a distinct aggressive lym- Ordonez NG. Broad-spectrum immunohistochemical epithelial mark- phoma with frequent gammadelta T-cell receptor expression. Am J ers: a review. Human Pathol. 2013;44(7):1195–1215. Surg Pathol. 2011;35(10):1557–1569. Ozcan A, Shen SS, Hamilton C, et al. PAX 8 expression in non-neoplastic Chu PG, Chen YY, Molina A, Arber DA, Weiss LM. Recurrent B-cell tissues, primary tumors, and metastatic tumors: a comprehensive neoplasms after Rituximab therapy: an immunophenotypic and immunohistochemical study. Mod Pathol. 2011;24(6):751–764. genotypic study. Leuk Lymphoma. 2002;43(12):2335–2341. Panarelli NC, Yantiss RK, Yeh MM, Liu Y, Chen YT. Tissue-specific Dong HY, Scadden DT, de Leval L, Tang Z, Isaacson PG, Harris NL. cadherin CDH17 is a useful marker of gastrointestinal adenocar- Plasmablastic lymphoma in HIV-positive patients: an aggressive cinomas with higher sensitivity than CDX2. Am J Clin Pathol. Epstein-Barr virus-associated extramedullary plasmacytic neo- 2012;138(2):211–222. plasm. Am J Surg Pathol. 2005;29(12):1633–1641. Saad RS, Silverman JF, Khalifa MA, Rowsell C. CDX2, cytokera- Dreyling M, Ferrero S, Vogt N, Klapper W. New paradigms in mantle tins 7 and 20 immunoreactivity in rectal adenocarcinoma. Appl cell lymphoma: is it time to risk-stratify treatment based on the Immunohistochem Mol Morphol. 2009;17(3):196–201. proliferative signature? Clin Cancer Res. 2014;20(20):5194–5206. Tacha D, Zhou D, Cheng L. Expression of PAX8 in normal and neo- Lai R, Weiss LM, Chang KL, Arber DA. Frequency of CD43 expres- plastic tissues: a comprehensive immunohistochemical study. Appl sion in non-Hodgkin lymphoma. A survey of 742 cases and further Immunohistochem Mol Morphol. 2011;19(4):293–299. characterization of rare CD43+ follicular lymphomas. Am J Clin Taniere P, Borghi-Scoazec G, Saurin JC, et al. Cytokeratin expression in Pahtol. 1999;111(4):488–494. adenocarcinomas of the esophagogastric junction: a comparative Menasce LP, Banerjee SS, Beckett E, Harris M. Extra-medullary study of adenocarcinomas of the distal esophagus and of the proxi- myeloid tumour (granulocytic sarcoma) is often misdiagnosed: a mal stomach. Am J Surg Pathol. 2002;26(9):1213–1221. study of 26 cases. Histopathology. 1999;34(5):391–398. Vang R, Gown AM, Wu LS, et al. Immunohistochemical expression O’Malley DP, Goldstein NS, Banks PM. The recognition and classifi- of CDX2 in primary ovarian mucinous tumors and metastatic cation of lymphoproliferative disorders of the gut. Hum Pathol. mucinous carcinomas involving the ovary: comparison with CK20 2014;45(5):899–916. and correlation with coordinate expression of CK7. Mod Pathol. Oschlies I, Burkhardt B, Chassagne-Clement C, d’Amore ES, Hansson 2006;19(11):1421–1428. U, Hebeda K, et al. Diagnosis and immunophenotype of 188 pedi- Wang NP, Zee S, Zarbo RJ, Bacchi CE, Gown AM. Coordinate expres- atric lymphoblastic lymphomas treated within a randomized pro- sion of cytokeratins 7 and 20 defined unique subsets of carcinomas. spective trial: experiences and preliminary recommendations from Appl Immunohistochem. 1995;3(2):99–107. the European childhood lymphoma pathology panel. Am J Surg WHO Classification of Tumours of Soft Tissue and Bone. 4th ed. Lyon: Pathol. 2011;35(6):836–844. IARC; 2013. Rubio-Tapia A, Murray JA. Classification and management of refrac- Winn B, Tavares R, Fanion J, et al. Differentiating the undifferenti- tory coeliac disease. Gut. 2010;59(4):547–557. ated: immunohistochemical profile of medullary carcinoma of the Ruzinova MB, Caron T, Rodig SJ. Altered subcellular localization of colon with an emphasis on intestinal differentiation. Hum Pathol. c-Myc protein identifies aggressive B-cell lymphomas harboring a 2009;40(3):398–404. c-MYC translocation. Am J Surg Pathol. 2010;34(6):882–891. Yemelyanova AV, Vang R, Judson K, Wu LS, Ronnett BM. Distinction Smith LB, Owens SR. Gastrointestinal lymphomas: entities and mimics. of primary and metastatic mucinous tumors involving the ovary: Arch Pathol Lab Med. 2012;136(8):865–870. analysis of size and laterality data by primary site with reevalua- Swerdlow SH, Yang WI, Zukerberg LR, Harris NL, Arnold A, Williams tion of an algorithm for tumor classification. Am J Surg Pathol. ME. Expression of cyclin D1 protein in centrocytic/mantle cell 2008;32(1):128–138. lymphomas with and without rearrangement of the BCL1/cyclin D1 gene. Hum Pathol. 1995;26(9):999–1004. Approach to Neuroendocrine Neoplasms Wu TT, Swerdlow SH, Locker J, Bahler D, Randhawa P, Yunis EJ, et al. Recurrent Epstein-Barr virus-associated lesions in organ trans- Bellizzi AM. Assigning site of origin in metastatic neuroendocrine neo- plant recipients. Hum Pathol. 1996;27(2):157–164. plasms: a clinically significant application of diagnostic immuno- histochemistry. Adv Anat Pathol. 2013;20(5):285–314. Fischler DF, Bauer TW, Tubbs RR. Tissue reactivity of anti-Leu19. Approach to Mesenchymal Tumors Histopathology. 1992;21(6):563–567. Maxwell JE, Sherman SK, Stashek KM, O’Dorisio TM, Bellizzi Arber DA, Tamayo R, Weiss LM. Paraffin section detection of the c-kit AM, Howe JR. A practical method to determine the site of gene product (CD117) in human tissues: value in the diagnosis of unknown primary in metastatic neuroendocrine tumors. Surgery. mast cell disorders. Hum Pathol. 1998;29(5):498–504. 2014;156(6):1359–1365; discussion 65–66. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, et al. Nuclear Rindi G, Arnold R, Bosman FT, Capella C, Klimstra DS, Kloppel G, et al. beta-catenin expression distinguishes deep fibromatosis from other Nomenclature and classification of neuroendocrine neoplams of the benign and malignant fibroblastic and myofibroblastic lesions. Am digestive system. In: Bosman FT, Carneiro F, Hruban RH, Theise J Surg Pathol. 2005;29(5):653–659. ND, eds. World Health Organization Classification of Tumours Carlson JW, Fletcher CD. Immunohistochemistry for beta-catenin in WHO Classification of Tumours of the Digestive System. 4th. ed. the differential diagnosis of spindle cell lesions: analysis of a series Lyon: IARC; 2010:13–14. and review of the literature. Histopathology. 2007;51(4):509–514. 374 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Coffin CM, Watterson J, Priest JR, Dehner LP. Extrapulmonary Medeiros F, Corless CL, Duensing A, et al. KIT-negative gastrointesti- inflammatory myofibroblastic tumor (inflammatory pseudotu- nal stromal tumors: proof of concept and therapeutic implications. mor). A clinicopathologic and immunohistochemical study of 84 Am J Surg Pathol. 2004;28(7):889–894. cases. Am J Surg Pathol. 1995;19(8):859–872. Miettinen M, Dow N, Lasota J, Sobin LH. A distinctive novel epi- Cook JR, Dehner LP, Collins MH, et al. Anaplastic lymphoma kinase theliomesenchymal biphasic tumor of the stomach in young (ALK) expression in the inflammatory myofibroblastic tumor: adults (“gastroblastoma”): a series of 3 cases. Am J Surg Pathol. a comparative immunohistochemical study. Am J Surg Pahtol. 2009;33(9):1370–1377. 2001;25(11):1364–1371. Miettinen M, Furlong M, Sarlomo-Rikala M, Burke A, Sobin LH, Deshpande A, Nelson D, Corless CL, Deshpande V, O’Brien MJ. Lasota J. Gastrointestinal stromal tumors, intramural leiomyo- Leiomyoma of the gastrointestinal tract with interstitial cells of mas, and leiomyosarcomas in the rectum and anus: a clinicopatho- Cajal: a mimic of gastrointestinal stromal tumor. Am J Surg Pathol. logic, immunohistochemical, and molecular genetic study of 144 2014;38(1):72–77. cases. Am J Surg Pathol. 2001;25(9):1121–1233. Doyle LA, Hornick JL, Fletcher CD. PEComa of the gastrointestinal Miettinen M, Kopczynski J, Makhlouf HR, et al. Gastrointestinal tract: clinicopathologic study of 35 cases with evaluation of prog- stromal tumors, intramural leiomyomas, and leiomyosarco- nostic parameters. Am J Surg Pathol. 2013;37(12):1769–1782. mas in the duodenum: a clinicopathologic, immunohistochemi- Espinosa I, Lee CH, Kim MK, et al. A novel monoclonal antibody cal, and molecular genetic study of 167 cases. Am J Surg Pathol. against DOG1 is a sensitive and specific marker for gastrointesti- 2003;27(5):625–641. nal stromal tumors. Am J Surg Pathol. 2008;32(2):210–218. Miettinen M, Makhlouf HR, Sobin LH, Lasota J. Plexiform fibromyx- Foo WC, Cruise MW, Wick MR, Hornick JL. Immunohistochemical oma: a distinctive benign gastric antral neoplasm not to be confused staining for TLE1 distinguishes synovial sarcoma from histologic with a myxoid GIST. Am J Surg Pathol. 2009;33(11):1624–1632. mimics. Am J Surg Pathol. 2011;135(6):839–844. Miettinen M, Paal E, Lasota J, Sobin LH. Gastrointestinal glomus Gibson JA, Hornick JL. Mucosal Schwann cell “hamartoma”: clinico- tumors: a clinicopathologic, immunohistochemical, and molec- pathologic study of 26 neural colorectal polyps distinct from neu- ular genetic study of 32 cases. Am J Surg Pathol. 2002;26(3): rofibromas and mucosal neuromas. Am J Surg Pathol. 2009;33(5): 301–311. 781–787. Miettinen M, Sarlomo-Rikala M, Sobin LH, Lasota J. Gastrointestinal Gill AJ, Chou A, Vilain R, et al. Immunohistochemistry for SDHB stromal tumors and leiomyosarcomas in the colon: a clinicopatho- divides gastrointestinal stromal tumors (GISTs) into 2 distinct logic, immunohistochemical, and molecular genetic study of 44 types. Am J Surg Pathol. 2010;34(5):636–644. cases. Am J Surg Pathol. 2000;24(10):1339–1352. Hollowood K, Stamp G, Zouvani I, Fletcher CD. Extranodal follicular Miettinen M, Virolainen M, Maarit Sarlomo R. Gastrointestinal stro- dendritic cell sarcoma of the gastrointestinal tract. Morphologic, mal tumors—value of CD34 antigen in their identification and immunohistochemical and ultrastructural analysis of two cases. separation from true leiomyomas and schwannomas. Am J Surg Am J Clin Pathol. 1995;103(1):90–97. Pathol. 1995;19(2):207–216. Hornick JL, Fletcher CD. Immunohistochemical staining for KIT Miettinen M, Wang ZF, Lasota J. DOG1 antibody in the differential (CD117) in soft tissue sarcomas is very limited in distribution. Am diagnosis of gastrointestinal stromal tumors: a study of 1840 J Clin Pathol. 2002;117(2):188–193. cases. Am J Surg Pathol. 2009;33(9):1401–1408. Hornick JL, Fletcher CD. Intestinal perineuriomas: clinicopathologic

Miettinen M, Wang ZF, Sarlomo-Rikala M, Osuch C, Rutkowski P, definition of a new anatomic subset in a series of 10 cases. Am J Lasota J. Succinate dehydrogenase-deficient GISTs: a clinicopath- Surg Pathol. 2005;29(7):859–865. ologic, immunohistochemical, and molecular genetic study of 66 Hornick JL, Fletcher CD. Validating immunohistochemical staining for gastric GISTs with predilection to young age. Am J Surg Pathol. KIT (CD117). Am J Clin Pathol. 2003;119(3):325–327. 2011;35(11):1712–1721. Iwata J, Fletcher CD. Immunohistochemical detection of cytokeratin Miettinen M. Immunoreactivity for cytokeratin and epithelial membrane and epithelial membrane antigen in leiomyosarcoma: a systematic antigen in leiomyosarcoma. Arch Pathol Lab Med. 1988;112(6): study of 100 cases. Pathol Int. 2000;50(1):7–14. 637–640. Killian JK, Miettinen M, Walker RL, Wang Y, Zhu YJ, Waterfall JJ, Miselli F, Millefanti C, Conca E, Negri T, Piacenza C, Pierotti MA, et al. Recurrent epimutation of SDHC in gastrointestinal stromal et al. PDGFRA immunostaining can help in the diagnosis of gastro- tumors. Sci Transl Med. 2014;6(268):268ra177. intestinal stromal tumors. Am J Surg Pathol. 2008;32(5):738–743. Lasota J, Wang ZF, Sobin LH, Miettinen M. Gain-of-function PDGFRA Montgomery E, Torbenson MS, Kaushal M, Fisher C, Abraham SC. mutations, earlier reported in gastrointestinal stromal tumors, are Beta-catenin immunohistochemistry separates mesenteric fibro- common in small intestinal inflammatory fibroid polyps. A study matosis from gastrointestinal stromal tumor and sclerosing mesen- of 60 cases. Mod Pathol. 2009;22(8):1049–1056. teritis. Am J Surg Pathol. 2002;26(10):1296–1301. Lee CH, Liang CW, Espinosa I. The utility of discovered on gastrointes- Montone KT, van Belle P, Elenitsas R, Elder DE. Proto-oncogene c-kit tinal stromal tumor 1 (DOG1) antibody in surgical pathology-the expression in malignant melanoma: protein loss with tumor pro- GIST of it. Adv Anat Pathol 2010;17(3):222–232. gression. Mod Pathol. 1997;10(9):939–944. Liu TC, Lin MT, Montgomery EA, Singhi AD. Inflammatory fibroid Parfitt JR, Rodriguez-Justo M, Feakins R, Novelli MR. Gastrointestinal polyps of the gastrointestinal tract: spectrum of clinical, mor- Kaposi’s sarcoma: CD117 expression and the potential for mis- phologic, and immunohistochemistry features. Am J Surg Pathol. diagnosis as gastrointestinal stromal tumour. Histopathology. 2013;37(4):586–592. 2008;52(7):816–823. Lopes LF, West RB, Bacchi LM, van de Rijn M, Bacchi CE. DOG1 for the Pileri SA, Grogan TM, Harris NL, et al. Tumours of histiocytes and diagnosis of gastrointestinal stromal tumor (GIST): Comparison accessory dendritic cells: an immunohistochemical approach to between 2 different antibodies. Appl Immunohistochem Mol classification from the International Lymphoma Study Group Morphol. 2010;18(4):333–337. based on 61 cases. Histopathology. 2002;41(1):1–29. Lucas DR, al-Abbadi M, Tabaczka P, Hamre MR, Weaver DW, Mott Prevot S, Bienvenu L, Vaillant JC, de Saint-Maur PP. Benign schwan- MJ. c-Kit expression in desmoid fibromatosis. Comparative immu- noma of the digestive tract: a clinicopathologic and immunohisto- nohistochemical evaluation of two commercial antibodies. Am J chemical study of five cases, including a case of esophageal tumor. Clin Pathol. 2003;119(3):339–345. Am J Surg Pathol. 1999;23(4):431–436. Makhlouf HR, Ahrens W, Agarwal B, et al. Synovial sarcoma of the Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, Miettinen M. CD117: stomach: a clinicopathologic, immunohistochemical, and molecu- a sensitive marker for gastrointestinal stromal tumors that is more lar genetic study of 10 cases. Am J Surg Pathol. 2008;32(2):275–281. specific than CD34. Mod Pathol. 1998;11(8):728–734. 13 Applications of Diagnostic Immunohistochemistry 375 Shin DH, Lee JH, Kang HJ, et al. Novel epitheliomesenchymal biphasic Distinguishing Poorly Cohesive Gastric Cancer stomach tumour (gastroblastoma) in a 9-year-old: morphological, ultrastructural and immunohistochemical findings. J Clin Pathol. From Metastatic Lobular Breast Cancer 2010;63(3):270–274. Chu PG, Weiss LM. Immunohistochemical characterization of signet- Singhi AD, Montgomery EA. Colorectal granular cell tumor: a clinicopath- ring cell carcinomas of the stomach, breast, and colon. Am J Clin ologic study of 26 cases. Am J Surg Pathol. 2010;34(8):1186–1192. Pathol. 2004;121(6):884–892. Stockman DL, Miettinen M, Suster S, Spagnolo D, Dominguez- Harris M, Howell A, Chrissohou M, Swindell RI, Hudson M, Sellwood Malagon H, Hornick JL, et al. Malignant gastrointestinal neu- RA. A comparison of the metastatic pattern of infiltrating lobu- roectodermal tumor: clinicopathologic, immunohistochemical, lar carcinoma and infiltrating duct carcinoma of the breast. Br J ultrastructural, and molecular analysis of 16 cases with a reap- Cancer. 1984;50(1):23–30. praisal of clear cell sarcoma-like tumors of the gastrointestinal McLemore EC, Pockaj BA, Reynolds C, et al. Breast cancer: presenta- tract. Am J Surg Pathol. 2012;36(6):857–868. tion and intervention in women with gastrointestinal metastasis Terry J, Saito T, Subramanian S, et al. TLE1 as a diagnostic immu- and carcinomatosis. Ann Surg Oncol. 2005;12(11):886–894. nohistochemical marker for synovial sarcoma emerging from Miettinen M, McCue PA, Sarlomo-Rikala M, et al. GATA3: a multi- gene expression profiling studies. Am J Surg Pathol. 2007;31(2): specific but potentially useful marker in surgical pathology: a sys- 240–246. tematic analysis of 2500 epithelial and nonepithelial tumors. Am J Wong NA, Campbell F, Shepherd NA. Abdominal monophasic Surg Pathol. 2014;38(1):13–22. synovial sarcoma is a morphological and immunohistochemi- Nazareno J, Taves D, Preiksaitis HG. Metastatic breast cancer to the cal mimic of gastrointestinal stromal tumour. Histopathology. gastrointestinal tract: a case series and review of the literature. 2015;66(7):974–981. World J Gastroenterol. 2006;12(38):6219–6224. Yamamoto H, Oda Y, Saito T, et al. p53 Mutation and MDM2 amplifi- Nguyen MD, Plasil B, Wen P, Frankel WL. Mucin profiles in signet-ring cation in inflammatory myofibroblastic tumours. Histopathology. cell carcinoma. Arch Pathol Lab Med. 2006;130(6):799–804. 2003;42(5):431–439. O’Connell FP, Wang HH, Odze RD. Utility of immunohistochemistry in distinguishing primary adenocarcinomas from metastatic breast carcinomas in the gastrointestinal tract. Arch Pathol Lab Med. Barrett’s Associated Dysplasia 2005;129(3):338–347. and Carcinoma Ordonez NG. Value of GATA3 immunostaining in tumor diagnosis: a review. Adv Anat Pathol. 2013;20(5):352–360. Baas IO, Mulder JW, Offerhaus GJ, Vogelstein B, Hamilton SR. An evaluation of six antibodies for immunohistochemistry of mutant p53 gene product in archival colorectal neoplasms. J Pathol. HER2 Assessment in Gastroesophageal 1994;172(1):5–12. Fels Elliott DR, Fitzgerald RC. Molecular markers for Barrett’s esoph- Adenocarcinoma agus and its progression to cancer. Curr Opin Gastroenterol. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et 2013;29(4):437–445. al. Trastuzumab in combination with chemotherapy versus chemo- Finlay CA, Hinds PW, Tan TH, Eliyahu D, Oren M, Levine AJ. therapy alone for treatment of HER2-positive advanced gastric or Activating mutations for transformation by p53 produce a gene gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, product that forms an hsc70-p53 complex with an altered half-life. randomised controlled trial. Lancet. 2010;376(9742):687–697. Mol Cell Biol. 1988;8(2):531–539. Bozzetti C, Negri FV, Lagrasta CA, Crafa P, Bassano C, Tamagnini I, Kastelein F, Biermann K, Steyerberg EW, et al. Aberrant p53 pro- et al. Comparison of HER2 status in primary and paired metastatic tein expression is associated with an increased risk of neoplas- sites of gastric carcinoma. Br J Cancer. 2011;104(9):1372–1376. tic progression in patients with Barrett’s oesophagus. Gut. Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system 2013;62:1676–1683. for gastric cancer: results from a validation study. Histopathology. Kaye PV, Haider SA, Ilyas M, et al. Barrett’s dysplasia and the Vienna 2008;52(7):797–805. classification: reproducibility, prediction of progression and Lee S, de Boer WB, Fermoyle S, Platten M, Kumarasinghe MP. Human impact of consensus reporting and p53 immunohistochemistry. epidermal growth factor receptor 2 testing in gastric carci- Histopathology. 2009;54(6):699–712. noma: issues related to heterogeneity in biopsies and resections. Kaye PV, Haider SA, James PD, et al. Novel staining pattern of p53 Histopathology. 2011;59(5):832–840. in Barrett’s dysplasia—the absent pattern. Histopathology. Perrone G, Amato M, Callea M, et al. HER2 amplification status in gastric 2010;57(6):933–935. and gastro-oesophageal junction cancer in routine clinical practice: which sample should be used? Histopathology. 2012;61(1):134–135. Ruschoff J, Dietel M, Baretton G, et al. HER2 diagnostics in gastric can- Poorly Differentiated Esophageal Carcinoma cer-guideline validation and development of standardized immu- DiMaio MA, Kwok S, Montgomery KD, Lowe AW, Pai RK. nohistochemical testing. Virchows Arch. 2010;457(3):299–307. Immunohistochemical panel for distinguishing esophageal ade- Ruschoff J, Hanna W, Bilous M, et al. HER2 testing in gastric cancer: a nocarcinoma from squamous cell carcinoma: a combination of practical approach. Mod Pathol. 2012;25(5):637–650. p63, cytokeratin 5/6, MUC5AC, and anterior gradient homolog 2 allows optimal subtyping. Hum Pathol. 2012;43(11):1799–1807. Huang Q, Wu H, Nie L, et al. Primary high-grade neuroendocrine Mismatch Repair Protein carcinoma of the esophagus: a clinicopathologic and immu- Immunohistochemistry nohistochemical study of 42 resection cases. Am J Surg Pathol. 2013;37(4):467–483. Bao F, Panarelli NC, Rennert H, Sherr DL, Yantiss RK. Neoadjuvant Long KB, Hornick JL. SOX2 is highly expressed in squamous therapy induces loss of MSH6 expression in colorectal carcinoma. cell carcinomas of the gastrointestinal tract. Hum Pathol. Am J Surg Pathol. 2010;34(12):1798–1804. 2009;40(12):1768–1773. Bellizzi AM, Frankel WL. Colorectal cancer due to deficiency in Pai RK, West RB. MOC-31 exhibits superior reactivity compared with DNA mismatch repair function: a review. Adv Anat Pathol. Ber-EP4 in invasive lobular and ductal carcinoma of the breast: a 2009;16(6):405–417. tissue microarray study. Appl Immunohistochem Mol Morphol. Boland CR. The mystery of mismatch repair deficiency: lynch or lynch- 2009;17(3):202–206. like? Gastroenterology. 2013;144(5):868–870. 376 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Carethers JM. Differentiating Lynch-like from Lynch syndrome. Affolter K, Samowitz W, Tripp S, Bronner MP. BRAF V600E muta- Gastroenterology. 2014;146(3):602–604. tion detection by immunohistochemistry in colorectal carcinoma. Engstrom PF, Arnoletti JP, Benson AB 3rd, et al. NCCN Clinical Genes Chromosomes Cancer. 2013;52(8):748–752. Practice Guidelines in Oncology: Colon Cancer. J Natl Compr Capper D, Voigt A, Bozukova G, et al. BRAF V600E-specific immu- Canc Netw. 2009;7(8):778–831. http://www.nccn.org/profession- nohistochemistry for the exclusion of Lynch syndrome in MSI-H als/physician_gls/pdf/colon.pdf colorectal cancer. Int J Cancer. 2013;133(7):1624–1630. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Kuan SF, Navina S, Cressman KL, Pai RK. Immunohistochemical detection Working Group. Recommendations from the EGAPP Working of BRAF V600E mutant protein using the VE1 antibody in colorectal Group: genetic testing strategies in newly diagnosed individuals carcinoma is highly concordant with molecular testing but requires with colorectal cancer aimed at reducing morbidity and mortality rigorous antibody optimization. Hum Pathol. 2014;45(3):464–472. from Lynch syndrome in relatives. Genet Med. 2009;11(1):35–41. Lasota J, Kowalik A, Wasag B, et al. Detection of the BRAF V600E Fadhil W, Field J, Cross G, Kaye P, Ilyas M. Immunostaining in the con- mutation in colon carcinoma: critical evaluation of the imunohis- text of loss mismatch repair function: interpretive confounders and tochemical approach. Am J Surg Pathol. 2014;38(9):1235–1241. cautionary tales! Histopathology. 2012;61(3):522–525. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutation-specific antibody Funkhouser WK, Jr., Lubin IM, Monzon FA, et al. Relevance, patho- detects mutant BRAFV600E protein expression in human colon genesis, and testing algorithm for mismatch repair-defective carcinomas. Cancer. 2013;119(15):2765–2770. colorectal carcinomas: a report of the association for molecular pathology. J Mol Diagn. 2012;14(2):91–103. Mismatch Repair Immunohistochemistry Hampel H. NCCN increases the emphasison genetic/familial high- risk assessment in colorectal cancer. J Natl Compr Canc Netw. in Colon Polyps 2014;12(5):829–831. http://www.nccn.org/professionals/physi- Halvarsson B, Lindblom A, Johansson L, Lagerstedt K, Nilbert M. Loss cian_gls/pdf/genetics_colon.pdf of mismatch repair protein immunostaining in colorectal adeno- Haraldsdottir S, Hampel H, Tomsic J, et al. Colon and endome- mas from patients with hereditary nonpolyposis colorectal cancer. trial cancers with mismatch repair deficiency can arise from Mod Pathol. 2005;18(8):1095–1101. somatic, rather than germline, mutations. Gastroenterology. Sheridan TB, Fenton H, Lewin MR, et al. Sessile serrated adenomas 2014;147(6):1308–1316. with low- and high-grade dysplasia and early carcinomas: an Hyde A, Fontaine D, Stuckless S, et al. A histology-based model for pre- immunohistochemical study of serrated lesions “caught in the act”. dicting microsatellite instability in colorectal cancers. Am J Surg Am J Clin Pathol. 2006;126(4):564–571. Pathol. 2010;34(12):1820–1829. Walsh MD, Buchanan DD, Pearson SA, et al. Immunohistochemical Jenkins MA, Hayashi S, O’Shea AM, et al. Pathology features in Bethesda testing of conventional adenomas for loss of expression of mis- guidelines predict colorectal cancer microsatellite instability: a pop- match repair proteins in Lynch syndrome mutation carriers: a case ulation-based study. Gastroenterology. 2007;133(1):48–56. series from the Australasian site of the colon cancer family registry. Mensenkamp AR, Vogelaar IP, van Zelst-Stams WA, et al. Somatic muta- Mod Pathol. 2012;25(5):722–30. tions in MLH1 and MSH2 are a frequent cause of mismatch-repair deficiency in Lynch syndrome-like tumors. Gastroenterology. 2014;146(3):643–646. Anal Squamous Intraepithelial Lesions/ Radu OM, Nikiforova MN, Farkas LM, Krasinskas AM. Challenging Intraepithelial Neoplasia (SIL/AIN) cases encountered in colorectal cancer screening for Lynch syndrome reveal novel findings: nucleolar MSH6 staining and impact of prior Bala R, Pinsky BA, Beck AH, Kong CS, Welton ML, Longacre TA. chemoradiation therapy. Hum Pathol. 2011;42(9):1247–1258. p16 is superior to ProEx C in identifying high-grade squamous Rodriguez-Soler M, Perez-Carbonell L, Guarinos C, et al. Risk of can- intraepithelial lesions (HSIL) of the

anal canal. Am J Surg Pathol. cer in cases of suspected lynch syndrome without germline muta- 2013;37(5):659–668. tion. Gastroenterology. 2013;144(5):926–932. Bean SM, Eltoum I, Horton DK, Whitlow L, Chhieng DC. Shia J, Stadler Z, Weiser MR, et al. Immunohistochemical staining for Immunohistochemical expression of p16 and Ki-67 correlates DNA mismatch repair proteins in intestinal tract carcinoma: how with degree of anal intraepithelial neoplasia. Am J Surg Pathol. reliable are biopsy samples? Am J Surg Pathol. 2011;35(3):447–454. 2007;31(4):555–561. Sourrouille I, Coulet F, Lefevre JH, et al. Somatic mosaicism and dou- Bernard JE, Butler MO, Sandweiss L, Weidner N. Anal intraepithelial ble somatic hits can lead to MSI colorectal tumors. Fam Cancer. neoplasia: correlation of grade with p16INK4a immunohistochem- 2013;12(1):27–33. istry and HPV in situ hybridization. Appl Immunohistochem Mol Morphol. 2008;16(3):215–220. Pirog EC, Quint KD, Yantiss RK. P16/CDKN2A and Ki-67 enhance the EPCAM Immunohistochemistry detection of anal intraepithelial neoplasia and condyloma and cor- Huth C, Kloor M, Voigt AY, et al. The molecular basis of EPCAM relate with human papillomavirus detection by polymerase chain expression loss in Lynch syndrome-associated tumors. Mod reaction. Am J Surg Pathol. 2010;34(10):1449–1455. Pathol. 2012;25(6):911–916. Walts AE, Lechago J, Bose S. P16 and Ki67 immunostaining is a use- Kloor M, Voigt AY, Schackert HK, Schirmacher P, von Knebel Doeberitz ful adjunct in the assessment of biopsies for HPV-associated anal M, Blaker H. Analysis of EPCAM protein expression in diagnos- intraepithelial neoplasia. Am J Surg Pathol. 2006;30(7):795–801. tics of Lynch syndrome. J Clin Oncol. 2011;29(2):223–227. Musulen E, Blanco I, Carrato C, et al. Usefulness of epithelial cell adhe- Approach to Tumors Presenting in a Pagetoid sion molecule expression in the algorithmic approach to Lynch syn- drome identification. Hum Pathol. 2013;44(3):412–416. Fashion in the Anal Canal Diaz de Leon E, Carcangiu ML, Prieto VG, et al. Extramammary Paget BRAF V600E Mutation-Specifi c disease is characterized by the consistent lack of estrogen and pro- Immunohistochemistry gesterone receptors but frequently expresses androgen receptor. Am J Clin Pathol. 2000;113(4):572–575. Adackapara CA, Sholl LM, Barletta JA, Hornick JL. Fogel BJ, Lai KK, Lindberg MR, Lamps LW, Quick CM. Pathologic Immunohistochemistry using the BRAF V600E mutation-specific Features Differentiating High-Grade Anal Squamous Intraepithelial monoclonal antibody VE1 is not a useful surrogate for genotyping in Lesions from Reactive Transition Zone. Mod Pathol. 2014;27(Suppl colorectal adenocarcinoma. Histopathology. 2013;63(2):187–193. 2):173A. 13 Applications of Diagnostic Immunohistochemistry 377 Goldblum JR, Hart WR. Perianal Paget’s disease: a histologic and immu- Sah SP, Kelly PJ, McManus DT, McCluggage WG. Diffuse CK7, CAM5.2 nohistochemical study of 11 cases with and without associated and BerEP4 positivity in pagetoid squamous cell carcinoma in situ rectal adenocarcinoma. Am J Surg Pathol. 1998;22(2):170–179. (pagetoid Bowen’s disease) of the perianal region: a mimic of extra- Goyal A, Zhang G, Yang B. Immunohistochemical Expression of mammary Paget’s disease. Histopathology. 2013;62(3):511–514. GATA3 in Primary Extramammary Paget Disease of the Vulva. Zeng HA, Cartun R, Ricci A, Jr. Potential diagnostic utility of CDX-2 Mod Pathol. 2014;27(Suppl 2):284A. immunophenotyping in extramammary Paget’s disease. Appl Nowak MA, Guerriere-Kovach P, Pathan A, Campbell TE, Deppisch Immunohistochem Mol Morphol. 2005;13(4):342–346. LM. Perianal Paget’s disease: distinguishing primary and second- Zhou L, Rao X, Jia L, et al. GATA3 is Expressed in Vulvar Paget’s ary lesions using immunohistochemical studies including gross Disease. Mod Pathol. 2014;27(Suppl 2):314A. cystic disease fluid protein-15 and cytokeratin 20 expression. Arch Pathol Lab Med. 1998;122(12):1077–1081. Ohnishi T, Watanabe S. The use of cytokeratins 7 and 20 in the diagno- Basaloid Squamous Cell Carcinoma Versus sis of primary and secondary extramammary Paget’s disease. Br J Basal Cell Carcinoma at the Anal Margin Dermatol. 2000;142(2):243–247. Ramalingam P, Hart WR, Goldblum JR. Cytokeratin subset immunos- Patil DT, Goldblum JR, Billings SD. Clinicopathological analysis of basal taining in rectal adenocarcinoma and normal anal glands. Arch cell carcinoma of the anal region and its distinction from basaloid Pathol Lab Med. 2001;125(8):1074–1077. squamous cell carcinoma. Mod Pathol. 2013;26(10):1382–1389. 14 Applications of Molecular Pathology RHONDA K. YANTISS INTRODUCTION They include a variety of growth factors, growth factor receptors, members of signal transduction pathways, and Gastrointestinal carcinomas account for more cancer- transcription factors that are normally produced in small related deaths than those of any other organ system. Recent quantities and rapidly degraded. Proto-oncogenes are clas- discoveries have uncovered key genetic, epigenetic, and post- sified as oncogenes when genetic alterations, such as point transcriptional regulatory mechanisms underlying their mutations, chromosomal translocations, and amplifica- development, some of which may be exploited for diagnos- tion, lead to dysregulation of their protein products and an tic and therapeutic purposes. Detection of tumor suppressor overall “gain of function.” Alterations of only one allele are or mismatch repair deficiencies may identify patients and generally sufficient to cause oncoprotein dysregulation. A family members with heritable cancer risk, whereas elucida- number of oncogenes have been implicated in the patho- tion of cellular signal transduction pathways continues to genesis of gastrointestinal malignancies and are routinely uncover proteins involved in cell survival and proliferation. evaluated in clinical situations, namely HER2, EGFR, Many of these molecules represent predictors of response to, KIT, KRAS, and BRAF. or potential targets of, directed medical therapies. Although a comprehensive discussion of the molecular Tumor Suppressor Genes abnormalities of all types of gastrointestinal malignancy is beyond the scope of this chapter, its purpose is to dis- Tumor suppressor genes are highly conserved across spe- cuss molecular changes that are important for patholo- cies and encode proteins that suppress transcription of gists to understand as they participate in the care of cancer proto-oncogenes. They can be inactivated through muta- patients, both in terms of diagnosis, risk assessment, and tions, translocations, loss of heterozygosity (LOH), and emerging treatment strategies (see Table 14.1). In addition, promoter hypermethylation that silences transcription. the majority of the prognostic and therapeutic molecular One functional allele is generally sufficient to regulate assays discussed in this chapter are now considered the proto-oncogenes, so dysregulation usually requires inac- standard of care for patient management. tivation of both tumor suppressor gene alleles. Most heri- table cancer syndromes result from germline mutations affecting tumor suppressor genes, in which case each cell harbors one dysfunctional allele. Inactivation of the sec- TERMINOLOGY ond allele may result from a second mutation, but more often reflects hypermethylation of its promoter region. Oncogenes The APC gene is one example of a tumor suppressor gene Proto-oncogenes encode regulatory proteins important to important to colorectal carcinogenesis; others include embryogenesis, proliferation, differentiation, and apoptosis. MUTYH, MLH1, PMS2, MSH2, and MSH6. 379 3 8 0 Neoplastic Gastrointestinal Pathology: An Illustrated Guide TABLE 14.1 Summary of Diagnostic, Therapeutic, and Predictive Molecular Tests in Gastrointestinal Neoplasia Molecular Feature Assessed Methodology Clinical Scenario HER2 In situ hybridization (ISH), f luorescence Adenocarcinoma of the esophagus, stomach, and in situ hybridization (FISH), gastroesophageal junction immunohistochemistry CDH1 Germline testing Suspected hereditary diffuse gastric cancer APC Germline testing Suspected familial adenomatous polyposis MUTYH Germline testing Suspected familial adenomatous polyposis, but APC wild-type SMAD4 Germline testing Suspected juvenile polyposis syndrome BMPR1A Germline testing Suspected juvenile polyposis syndrome LKB1/STK11 Germline testing Suspected Peutz–Jeghers polyposis syndrome PTEN Germline testing Suspected PTEN hamartoma tumor syndrome SDHB and SDHD Germline testing Suspected PTEN hamartoma tumor syndrome, but wild-type PTEN in some reports DNA Mismatch Repair Microsatellite instability (MSI) Suspected Lynch syndrome Mechanisms Immunohistochemistry for MLH1, MSH2, PMS2, MSH6 Germline MLH1, MSH2, PMS2, or MSH6 based on results of immunohistochemistry BRAF Mutational testing in tumor Suspected Lynch syndrome; loss of MLH1/PMS2 with BRAF mutation is virtually diagnostic of sporadic tumor KRAS Mutational testing in tumor Management of advanced colorectal carcinoma KIT Mutational testing in tumor Gastrointestinal stromal tumor PDGFRA Mutational testing in tumor Gastrointestinal stromal tumor with wild-type KIT SDHA and SDHB Immunohistochemistry mutational Gastrointestinal stromal tumor; immunohistochemistry testing in tumor based on results of used to screen for tumors with succinate immunohistochemistry dehydrogenase deficiency Mutations occur in KIT/PDGFRA wild-type tumors MicroRNAs developed techniques to introduce deficient microRNAs into tumor cells. Clinical applications of microRNA tech- MicroRNAs comprise a recently recognized class of mol- nology are at an early stage of development and none are ecules involved in the post-transcriptional regulation of currently utilized in the management of cancer patients. protein expression. These short, noncoding sequences are part of complexes that bind to mRNA strands and prevent their translation. MicroRNAs may have either KEY MOLECULAR FEATURES OF growth promoting or tumor suppressor activity. Some GASTROINTESTINAL MALIGNANCIES microRNAs normally suppress translation of oncogenic mRNA transcripts, thus negatively regulating cell prolif- Barrett Esophagus and Associated eration. Decreased expression of these microRNA species Adenocarcinoma promotes cell proliferation by removing their inhibitory Overview effects. Conversely, abnormally increased expression of microRNAs that prevent translation of tumor suppressor Adenocarcinomas of the distal esophagus and proximal gene transcripts further diminish their capacity to regulate stomach (ie, cardia) share similar epidemiologic and histo- cell growth. Data from preliminary studies indicate that logic features (see also Chapters 7 and 8). Most are associ- microRNAs may be of predictive value or represent attrac- ated with gastroesophageal reflux disease and intestinal tive therapeutic targets among gastrointestinal malignan- metaplasia, although some tumors arising in the cardia cies. Several agents that inhibit microRNAs show some develop in association with Helicobacter pylori related promise as therapeutic agents in vitro and in animal stud- pangastritis and display epidemiologic features typical of ies. Antisense oligonucleotides comprise one class of such distal gastric carcinomas. Metaplastic epithelium in this molecules that competitively inhibit microRNAs, whereas location is genetically unstable and prone to accumulate microRNA sponges consist of synthetic polynucleotides molecular changes, even in the absence of dysplasia. The that contain multiple tandem sequences complementary to acquisition of genetic events confers a growth advantage specific miRNAs. These sponges act as decoy transcripts to clonal subpopulations, leading to the evolution of low- to sequester miRNA complexes and prevent their bind- grade dysplasia, high-grade dysplasia, and, ultimately, ing to mRNA transcripts. Some investigators have also invasive carcinoma. The overall cancer risk of Barrett 14 Applications of Molecular Pathology 3 81 esophagus is relatively low (less than 2%), but increases the same (homodimerization) or different (heterodimer- among patients with low-grade (10%) and high-grade ization) class, followed by internalization of the recep- dysplasia (25%). Cancer risk among patients with intes- tor and autophosphorylation of tyrosine kinase domains. tinal metaplasia limited to the gastric cardia is even lower Autophosphorylation of internal domains initiates intra- than that of Barrett esophagus, presumably reflecting the cellular signaling of key regulatory pathways, includ- smaller surface area affected by metaplasia. ing the Ras/Raf/mitogen-activated protein kinase, PI3K/ AKT, and signal transducers and activators of transcrip- Molecular Alterations tion (STAT) signaling pathways, which promote cell pro- liferation and prolong cell survival. Approximately 30% Specific molecular changes in nondysplastic epithelium to 60% of esophageal and gastroesophageal junctional may predict subsequent cancer risk among patients with adenocarcinomas show amplification of EGFR, which intestinal metaplasia of the esophagus and gastroesopha- is associated with a negative clinical outcome (see also geal junction. Abnormal DNA content and LOH at 17p Chapter 13). Amplification of chromosome 17q21 is seen (TP53) and 9p, as well as overexpression of cyclins D1 in 25% to 30% of cases. This region contains ERBB2, and E, are strongly associated with the development of which encodes HER2. Importantly, HER2 is the preferred esophageal adenocarcinoma. Messenger RNA levels of heterodimerization partner of other members of the fam- inducible nitric oxide synthase progressively increase ily and, when present in high concentrations, it can spon- among patients with gastroesophageal reflux disease, taneously dimerize in the absence of ligand binding. For Barrett esophagus, and esophageal adenocarcinoma. This these reasons, HER2 represents the most important target enzyme facilitates production of free nitric oxide radicals, of directed therapy in the tyrosine kinase receptor family. which generate reactive oxygen and nitrogen species that promote DNA alterations, post-translational modification of TP53, and oxidization of thiols, all of which contribute Targeted Molecular Therapies to carcinogenesis. Finally, the NF (nuclear factor)-κB sig- Cetuximab is a chimeric monoclonal antibody that binds naling cascade plays an important role in stress responses, to EGFR and inhibits ligand-induced dimerization of the immune cell activation, apoptosis, proliferation, and dif- receptor. Although initial data suggested potential benefits ferentiation. Increased NF-κB levels also show an inverse of cetuximab in the management of advanced stage esoph- correlation with a robust pathologic response to neoadju- ageal and gastroesophageal junctional adenocarcinomas, vant therapy, suggesting that this marker may be of pre- several phase II and III trials have yielded only modest dictive

value among patients with adenocarcinomas of the results when the drug is used in combination with conven- distal esophagus and gastroesophageal junction. tional chemotherapy. Cetuximab as a second-line, single In addition to the above-mentioned features, most agent has essentially no efficacy when used in the man- adenocarcinomas of the esophagus and gastroesopha- agement of patients with metastatic disease and, unlike geal junction show alterations in tumor suppressor genes, carcinomas of the colorectum, the likelihood of response oncogenes, growth factor receptors, or enzymes involved to inhibitor therapy is not clearly linked to the pres- in cell signaling. Common mechanisms of dysregulation ence of mutations in KRAS, BRAF, PI3KCA, or PTEN. include chromosomal instability, amplification, and epi- Trastuzumab is a humanized monoclonal antibody that genetic modification of key genes. Alterations of TP53 binds the HER2 extracellular domain and prevents acti- resulting from mutations and LOH are detected in up to vation of its intracellular tyrosine kinase. Data from sev- 80% of cases. Amplification of chromosomes 7p (EGFR), eral studies clearly show that this agent prolongs survival 8q (MYC), and 17q (ERBB2) are detected in more than among patients with advanced adenocarcinomas of the 40% of cases. Hypermethylation, or LOH, involving distal esophagus and gastroesophageal junction that dis- CDKN2A/p16 is detected in up to 80% of cases and play moderate to strong basolateral or membranous stain- occurs early in cancer development. Promoter methyla- ing for HER2 by immunohistochemistry (see also Chapter tion of CDH1, APC, MGMT, and TMEFF2/HPP1 is also 13), or show gene amplification by in situ hybridization common. (Figure 14.1). Other growth factor receptor inhibitors tar- Epidermal growth factor receptor (EGFR) is one geting EGFR (eg, matuzumab, panitumumab, and nimo- member of the HER family of tyrosine kinase receptors tuzumab) or showing anti-vascular endothelial growth and has been implicated in the pathogenesis of several factor (VEGF) activity (eg, bevacizumab, sunitinib, and types of gastrointestinal cancer. This receptor has an sorafenib) are currently under investigation. extracellular ligand binding domain, a transmembrane Tyrosine kinase inhibitors represent a second class of region, an intracellular domain with tyrosine kinase activ- agents that showed promise in cell line studies and early ity, and a tail of tyrosine residues responsible for down- clinical trials of patients with carcinoma of the upper stream signaling. Ligand binding induces dimerization gastrointestinal tract. Agents under investigation include of the receptor with another tyrosine kinase receptor of gefitinib, erlotinib, and lapatinib, an orally administered 3 8 2 Neoplastic Gastrointestinal Pathology: An Illustrated Guide (A) (B) FIGURE 14.1 Immunohistochemical staining for HER2 is often patchy, and this pattern is considered a positive result. This case was associated with amplification by in situ hybridization. Discontinuous, predominantly basolateral staining of the cell membrane is present in approximately 50% of the tumor cells (A). Amplification of ERBB2 results in overexpression of HER2 receptor. Several tumor cells contain multiple copies denoted by red signals in this in situ hybridization assay (arrow). The green signals represent chromosome 17 centromeric signals (B, courtesy of Dr. Shivakumar Subramaniyam). small molecule tyrosine kinase inhibitor that targets Hypermethylation of MGMT, a DNA repair gene, is asso- HER2 and EGFR. Unfortunately, most compounds evalu- ciated with infection by CagA-positive strains of H. pylori ated to date have shown disappointing results when used and is a common feature of gastric carcinomas. to treat patients with esophageal and gastroesophageal junctional adenocarcinomas. Molecular Alterations Gastric Adenocarcinoma The molecular features of gastric carcinoma are variable and related to tumor morphology. Intestinal-type carci- Overview nomas generally display molecular alterations similar to Gastric adenocarcinoma is a major cause of global those of colorectal carcinomas, namely LOH or mutations cancer-related morbidity and mortality. Most (greater than affecting APC (30%–40%), DCC (60%), KRAS (up to 80%) cases are sporadic and develop in patients with long- 30%), and TP53 (25–40%), whereas abnormalities affect- standing chronic gastritis and mucosal atrophy due to a ing these genes are detected in less than 2% of diffuse-type variety of etiologies, including chronic H. pylori infection, carcinomas. Epigenetic alterations and promoter methyla- autoimmune gastritis, Ménétrier disease, and long-stand- tion are also more common among intestinal-type gastric ing chemical gastropathy, especially in patients who have adenocarcinomas. Up to one-third of tumors show meth- undergone distal gastrectomy and a Billroth II procedure ylation of the CDKN2A promoter and nearly two-thirds (see also Chapter 8). Strains of H. pylori that express vacu- show hypermethylation of the retinoic acid receptor-β olating cytotoxin (VacA) and cytotoxin-associated antigen (RARB). High-frequency microsatellite instability (MSI- A (CagA) are more closely linked to cancer risk because H) is more frequently encountered in intestinal-type car- they elicit a robust inflammatory response and promote cinomas (15%–40%) compared to diffuse-type tumors carcinogenesis through a variety of mechanisms. Infection (5%–10%). Virtually all sporadic carcinomas with MSI-H promotes expression of inducible nitric oxide synthetase develop via hypermethylation of the MLH1 promoter, and oxidative stress in epithelial stem cells, resulting in similar to MSI-H colon cancers, although gastric carci- elaboration of free radicals and active oxygen species that nomas may also represent a manifestation of hereditary cause DNA damage. The organism also facilitates bind- nonpolyposis colon cancer (HNPCC). Morphologic fea- ing of activator protein-1, c-Jun, and c-Fos to the promoter tures linked to MSI-H gastric adenocarcinomas include region of EGFR, indirectly activates NF-kB-mediated tumor heterogeneity, abundant lymphoid stroma with transcription, and induces TNF (tumor necrosis factor)-α. intraepithelial lymphocytes (ie, medullary carcinoma), 14 Applications of Molecular Pathology 3 8 3 and mucinous differentiation. Some gastric adenocarcino- of cases), whereas the remaining 20%–25% of cases are mas, particularly those of intestinal-type, show increased due to missense mutations. Unlike sporadic diffuse-type expression of receptor kinases. Up to 20% of cases show gastric cancers that show clustering of mutations around increased EGFR copy number by in situ hybridization, exons 7 and 8, germline mutations can occur over a large reflecting gene amplification or polysomy of chromosome part of the gene of the affected allele. Thus, screening of 7. Approximately 25% of intestinal-type tumors over- all 16 exons is generally necessary when a potential diag- express HER2 compared to 5% of diffuse-type carcino- nosis of hereditary diffuse gastric cancer is considered. mas and 10% of tumors containing intestinal and diffuse Of note, both familial and sporadic diffuse gastric can- areas. cers may show decreased, or absent, E-cadherin immu- The molecular features of purely diffuse-type carci- noexpression, so this technique cannot be reliably used to nomas have not been as extensively studied as those with identify patients with germline mutations. Patients with an intestinal phenotype, although these tumors do show suspected hereditary diffuse gastric cancer require evalu- some distinct alterations. More than 50% of diffuse-type ation of peripheral blood DNA, usually in the form of gastric adenocarcinomas show diminished E-cadherin direct sequencing. expression, reflecting abnormalities of CDH1 located on chromosome 16q22. This calcium-dependent transmem- brane glycoprotein normally interacts with either β-catenin Targeted Molecular Therapies or γ-catenin to facilitate cell–cell adhesion. Slightly more As is the case in adenocarcinomas of the esophagus and than half of sporadic tumors harbor CDH1 mutations, or gastroesophageal junction, trastuzumab improves survival LOH, in a single allele and show promoter hypermethyl- of patients with advanced gastric cancers that overexpress ation of the other allele, indicating that hypermethylation HER2 when used in combination with conventional che- represents the “second hit” responsible for completely sup- motherapy. Although data from the ToGA trial suggested pressed E-cadherin expression in these tumors. that immunohistochemical overexpression of HER2 cor- Epstein–Barr virus (EBV) is important to the develop- relates with the response to trastuzumab better than in ment of up to 10% of gastric cancers, particularly those situ hybridization, several studies since have shown com- containing abundant lymphoid stroma or developing in parable predictive values of these assays and many labo- the remnant stomach following a Billroth II procedure. ratories now use either immunohistochemistry, in situ The background mucosa is frequently inflamed or atro- hybridization, or both to determine whether patients with phic, suggesting a role of chronic injury in the development advanced gastric cancer should receive trastuzumab (see of EBV-related gastric cancers. Mechanisms underlying also Chapter 13). virally induced oncogenesis are not entirely clear, but such Ramucirumab is a fully human monoclonal anti- tumors often show epigenetic DNA hypermethylation body directed against vascular endothelial growth fac- of promoter regions in tumor suppressor genes, such as tor receptor 2 (VEGFR2). It functions as a receptor MGMT, MINT2, PTEN, and RASSF1A. These tumors antagonist to competitively inhibit binding of VEGF also show loss of cell cycle regulation with increased cyclin and prevent angiogenesis and proliferation. This agent D1 and decreased p16 expression. was recently approved as a second-line therapy in the management of advanced gastric cancer. It is the first biologic treatment shown to be of survival benefit when Hereditary Diffuse Gastric Cancer administered as a single agent to patients with meta- Hereditary diffuse gastric cancer is an autosomal domi- static gastric cancer. nant cancer syndrome characterized by signet ring cell Unfortunately, most other targeted molecular thera- carcinoma of the stomach, often in combination with pies analyzed to date are of limited value in the manage- lobular breast carcinoma. Families with the syndrome ment of gastric carcinoma, or are in preliminary stages are defined by the presence of two or more documented of development. Cetuximab, an EGFR inhibitor, was cases of diffuse gastric cancer in first- or second-degree initially reported to improve survival in patients with relatives, at least one of whom is diagnosed before age 50; advanced gastric cancer, but the efficacy of this agent has or three or more cases of diffuse gastric cancer in first- or not borne out in recent phase III clinical trials. Tyrosine second-degree relatives, regardless of age. The presence of kinase inhibitors have minimal activity against gastric either of these features, a diagnosis of diffuse gastric can- carcinomas. Several other agents show promising antitu- cer at a young age (less than 40 years), or a family history mor effects in preclinical studies, including inhibitors of of both lobular breast cancer and diffuse gastric cancer insulin-like growth factor-1 receptor, fibroblast growth should prompt evaluation for germline CDH1 mutations, factor receptor, and c-Met signaling. Histone deacetylase as they underlie a substantial proportion (30%–40%) of inhibitors represent potentially valuable agents, as they cases. Truncating mutations that produce a premature can theoretically promote re-expression of previously stop codon are most commonly identified (75%–80% silenced tumor suppressor genes. 3 8 4 Neoplastic Gastrointestinal Pathology: An Illustrated Guide Colorectal Cancer is normally expressed in nonproliferating colorectal epi- thelium and is essential for regulating cell growth and Overview differentiation. Loss of APC affects the Wnt signaling Colorectal cancer is the third most common cause of cancer- pathway, which is an evolutionarily conserved mecha- related mortality in the United States. It is now largely con- nism important to colorectal neoplasia. In the absence of sidered to be a genetic disease driven by a complex array of Wnt signaling, the APC–axin–GSK3ß cytoplasmic com- genetic and epigenetic alterations. Early mutations in cancer plex sequesters β-catenin and targets it for destruction. cells affect genomic stability and propagate additional alter- However, activation of Wnt signaling interferes with the ations that facilitate cell proliferation. Two major types of APC–axin–GSK3ß cytoplasmic complex and prevents genomic instability occur in colorectal carcinoma: chromo- phosphorylation and ubiquitination of β-catenin, result- somal instability and microsatellite instability (MSI). Both of ing in its accumulation within the cytoplasm. Excess these mechanisms occur in sporadic colorectal carcinomas β-catenin translocates to the nucleus where it forms a as well as in association with familial syndromes, namely complex with T cell factor and lymphoid enhancer factor familial adenomatous polyposis and Lynch syndrome that promotes expression of several important cell cycle (hereditary nonpolyposis colorectal cancer), respectively (see regulating genes. Thus, APC negatively regulates Wnt sig- also Chapters 6 and 11). Study of these familial cancers has naling via participation in the APC–axin–GSK3ß destruc- facilitated our understanding of the mechanisms underlying tion complex that promotes β-catenin degradation. sporadic tumors and aided recognition of several clinically The antitumorigenic effects of APC are not limited to important molecular aberrations. Mutations in tumor sup- β-catenin degradation. It also acts on promoters of Wnt- pressor and DNA repair genes aid identification of patients responsive genes and shuttles β-catenin out of the nucleus and family members with heritable cancer risk, whereas oth- for destruction. It binds microtubules and F-actin to nega- ers have

prognostic and therapeutic implications. tively regulate cell cycling and direct cell migration, and interferes with microtubule dynamics that directly affect Molecular Alterations mitosis. Cells containing mutant APC are predisposed to mitotic errors and aneuploidy due to defects in mitotic APC/Wnt Signaling and Chromosomal Instability spindles. Inactivation of APC also promotes chromosomal Most sporadic colorectal carcinomas, and all tumors that alterations involving KRAS2 (12p12), chromosome 18q, develop in association with familial adenomatous polypo- and TP53 (17q13). sis, display chromosomal instability and biallelic adeno- matous polyposis coli (APC) inactivation. Mutations Microsatellite Instability in APC are either nonsense mutations that introduce a premature stop codon or frameshift mutations resulting Microsatellite instability is a term used to describe expan- from insertions or deletions. Tumors with chromosomal sion or contraction of short nucleotide repeats (microsatel- instability are characterized by gains and losses of large lites), which are prone to replication errors because DNA amounts of genetic material (ie, LOH) that results in polymerase frequently slips over repetitive sequences. karyotypic variability among tumor cells. Patients with Slippage of the DNA strand creates insertion–deletion familial adenomatous polyposis generally have at least one loops and single base pair mismatches that are normally mutation between codons 1250 and 1450. Germline muta- recognized and corrected by the mismatch repair system. tions in this region are followed by mutations in the sec- Dysfunctional mismatch repair mechanisms result in a fail- ond allele that result in complete loss of the gene, whereas ure to correct these errors and their propagation during the germline mutations outside this region are associated second round of replication. Both insertion–deletion loops with somatic mutations between codons 1250 and 1450. and single base pair mismatches alter the lengths of micro- Selection for at least one mutation in this region ensures satellites in the DNA of tumor cells compared to those of that mutant APC retains some capacity to bind β-catenin non-neoplastic tissues. Microsatellites are more numerous and provides enough Wnt/β-catenin signaling to promote in noncoding regions of the genome, although alterations in tumor formation. Importantly, some patients with a clini- those that occur in coding regions can lead to downstream cal phenotype suggesting familial adenomatous polyposis nonsense mutations. Single base pair mismatches result in have biallelic inactivating mutations of MUTYH, a base point mutations and insertion–deletion loops cause frame- excision repair gene, rather than germline APC mutations. shift mutations in the defective daughter strand. This gene is located at 1p32-34 and contains mutational MSI is seen in two clinical contexts. This mechanism hot spots at Y165C and G382D. Patients with inactive accounts for nearly all tumors associated with Lynch syn- MUTYH acquire somatic APC mutations as a conse- drome (see also Chapters 6 and 11) as well as the devel- quence of impaired base excision repair function. opment of 10% to 15% of sporadic colorectal cancers. Inactivation of APC occurs early in the progression of Tumors that occur in association with Lynch syndrome adenomas to adenocarcinomas. The APC protein product generally result from a germline mutation in one of four 14 Applications of Molecular Pathology 3 8 5 mismatch repair genes: MLH1, MSH2, MSH6, or PMS2. Results of immunohistochemistry for mismatch repair Sporadic microsatellite unstable tumors usually display proteins correlate well with those of MSI testing by PCR of acquired hypermethylation of the MLH1 promoter, which microsatellite repeats. Colorectal cancers normally display suppresses its transcription, although biallelic inactivation nuclear staining of all four mismatch repair proteins, whereas of any mismatch repair gene can occur via a combination loss of staining of one or more of these proteins implies mis- of somatic mutations and methylation. match repair deficiency (see also Chapters 11 and 13). The Tumors are evaluated for MSI using polymerase chain pattern of staining reflects the tendency of mismatch repair reaction (PCR) to amplify a panel of five microsatellite proteins to form heterodimers. Tumors deficient in MLH1 repeats. The Bethesda panel consists of two mononucleotide show loss of MLH1 and PMS2 staining because these pro- repeats (eg, AAAAAAAAA) and three dinucleotide repeats teins form a heterodimer that stabilizes PMS2. Similarly, (eg, CACACACACA). Tumors are classified as MSI-H if tumors deficient in MSH2 show loss of MSH2 and MSH6 two or more repeats showed instability, MSS (microsatellite because the MSH2/MSH6 heterodimer stabilizes MSH6. stable) if no repeats are unstable, and MSI-low if one repeat In contrast, both MLH1 and MSH2 staining are preserved is unstable. The biologic significance of the latter category when either PMS2 or MSH6 is lost, probably reflecting the is controversial, but is probably irrelevant to the detec- capacities of MLH1 and MSH2 to bind other partners. tion of Lynch syndrome. As it turns out, mononucleotide There are several caveats to the interpretation of repeat instability is more sensitive and specific for mismatch immunohistochemical stains for mismatch repair pro- repair deficiency than instability at dinucleotide repeats teins. Tumor staining should only be evaluated in areas and, thus, commercially available panels that employ five that show staining of internal control cells, such as lym- mononucleotide repeats without any dinucleotide repeats phocytes or fibroblasts; negative tumor staining in areas have increasingly replaced the Bethesda panel in clinical without internal control staining is not interpretable. One practice. Instability at two or more mononucleotide repeats important exception to this rule is the patient with consti- is classified as MSI-H and no instability is considered to tutional mismatch repair deficiency. Tumors from these be MSS (Figure 14.2). Instability in one repeat is classified patients show an absence of staining for mismatch repair as indeterminate rather than MSI-low because this finding proteins in both tumor and normal tissues. However, the may reflect underlying mismatch repair deficiency. constellation of clinical features is an important clue to FIGURE 14.2 Microsatellite instability is present in the BAT-25 mononucleotide repeat. DNA from a colonic adenocarcinoma with MSI shows left-shifted peaks in this microsatellite relative to the non-neoplastic tissue from the same patient (courtesy of Dr. Wade Samowitz). 3 8 6 Neoplastic Gastrointestinal Pathology: An Illustrated Guide the underlying diagnosis. Missense changes in a protein Tumors that show DNA hypermethylation at multi- that destroy its function may have preserved antigenicity ple promoters are classified as showing the CpG Island that leads to a false negative immunohistochemical result. Methylator Phenotype (CIMP). They frequently display Abnormal immunohistochemical staining is usually mani- MSI-H and wild-type TP53, as well as BRAF muta- fest as complete loss of nuclear staining, although weak tions and methylation of MINT1, MLH1, RIZ1, and nuclear staining, rather than complete loss, may rarely TIMP3. Higher levels of DNA methylation are associated reflect the presence of a germline mutation. Combined with BRAF mutations compared with KRAS mutations loss of MLH1 and PMS2 staining usually reflects defi- among colorectal cancers, whereas tumors wild-type for cient MLH1, which may reflect a germline MLH1 both BRAF and KRAS show essentially no methylation. mutation (Lynch syndrome) or acquired MLH1 hyper- Colorectal carcinomas with CIMP tend to occur in the methylation in sporadic tumors. Distinction between proximal colons of older women and are associated with Lynch-associated tumors with defective MLH1 and spo- a history of cigarette smoking. They frequently harbor radic cancers is facilitated by assessment for the presence BRAF mutations and show methylation of MLH1 result- of the Val600Glu (V600E) mutation in BRAF and MLH1 ing in MSI-H in up to 70% of cases. methylation. More than 50% of sporadic MSI-H colorec- tal cancers harbor BRAF mutations. This alteration has Targeted Molecular Therapies not been described in Lynch-related tumors with MLH1 Cetuximab and panitumumab are monoclonal antibodies mutations, but rarely occur in those with PMS2 mutations against EGFR that are used to treat metastatic colorectal that show a different immunohistochemical staining pro- cancer. Drug efficacy is unrelated to EGFR expression by file (ie, isolated loss of PMS2 staining). Hypermethylated immunohistochemistry and EGFR mutations are infre- MLH1 is characteristic of sporadic MSI-H tumors, but quently encountered in colorectal carcinoma. Thus, evalu- may also be seen in occasional Lynch-associated tumors, ation of colorectal cancers for EGFR expression is of no so this feature is less reliable than a BRAF mutation in clinical value at this time. However, mutations affecting distinguishing sporadic and syndromic carcinomas. genes in signaling pathways downstream of EGFR predict Other types of genetic alterations may underlie rare a lack of response to EGFR antagonists. Oncogenic activa- cases of Lynch syndrome. Some patients with pheno- tion of these downstream genes leads to activation of cell typic Lynch syndrome have heritable epigenetic inactiva- signaling despite pharmacologic inhibition of EGFR bind- tion of MLH1, or MLH1 constitutional epimutation, in ing to its ligand. For this reason, these agents are not effec- which case the germline defect is promoter methylation of tive against tumors that harbor mutations affecting cell MLH1. Other patients with loss of MSH2 and MSH6 by signaling downstream of EGFR. For example, colorectal immunohistochemistry have germline deletions affecting cancers with mutations in codons 12 and 13 of KRAS show the polyadenylation site located in exon 9 of epithelial cell a lack of, or diminished, response to cetuximab and pani- adhesion molecule (EPCAM), which is located upstream tumumab. These monoclonal antibodies are very expensive of MSH2. Deletion of this region results in loss of the therapeutic agents and may cause severe toxicities, so it is EPCAM stop codon and subsequent CpG methylation understandable that the Food and Drug Administration in MSH2 or formation of EPCAM–MSH2 fusion tran- now mandates mutational evaluation of these KRAS codons scripts, both of which abrogate the function of MSH2. before therapy with such agents is initiated (Figure 14.3). Several other types of mutation may abrogate the effi- The CpG Island Methylator Phenotype cacy of cetuximab and panitumumab. Oncogenic KRAS There are several types of epigenetic alteration that regu- mutations in codons 61, 117, and 146 decrease the effi- late gene expression. These include DNA hypermethyl- cacy of cetuximab and similar agents, as do mutations ation, DNA hypomethylation, post-translational histone in NRAS, another member of the Ras family. Mutations modification, and chromatin looping. Methyltransferases in BRAF would also be predicted to activate this path- add methyl groups to the carbon 5 position of cytosine way independent of EGFR and certainly BRAF-mutated residues located 5´ to guanine. Subsequent methyl cyto- MSS colorectal cancers are associated with decreased sur- sines are clustered in regions rich in cytosine (C) and gua- vival, but it is not clear whether this observation reflects a nine (G) dinucleotides (ie, CpG islands) that are present in lack of response to EGFR inhibitor therapy or the inher- the promoter regions of up to 50% of mammalian genes. ently aggressive nature of BRAF-mutated MSS cancers. Promoter methylation in this fashion suppresses gene tran- Mutations in exon 20 of PIK3CA affect the PIK3CA– scription by inhibiting binding of transcription factors, AKT–mTor pathway downstream of EGFR and are asso- affecting histone acetylation, and altering conformations ciated with a lack of responsiveness to cetuximab. Loss to effectively block access of transcriptional machinery to of PTEN, an inhibitor of PIK3CA, also serves to activate the gene. Epigenetic hypermethylation of genes prevents the PIK3CA–AKT–mTor pathway and is associated with their transcription and may account for the first, second, a lack of responsiveness to EGFR antagonists. Cetuximab or both hits in silencing tumor suppressor genes. and similar agents are most likely to be beneficial among 14 Applications of Molecular Pathology 3 87 A: 1% C: 0% G: 99% A: 1% T: 0% G: 99% 75 50 Normal 25 0 -25 E S T A C G A C T C A G A T G C G T A G 5 10 A:35% C:1% GATGGC G: 64% A: 2% T: 0% G: 98% 100 75 c.35G>A, 50 p.G12D 25 0 E S T A C G A C T C A G A T G C G T A G 5 10 FIGURE 14.3 Pyrosequencing detects a 35G>A p.G12D mutation in KRAS in a colorectal carcinoma. Results of DNA analysis of the normal tissue are provided for comparison (courtesy of Dr. Wade Samowitz). patients with tumors that are wild-type KRAS, wild-type kinases. Most occur in exon 11, promoting constitu- BRAF, wild-type PIK3CA, and maintained PTEN expres- tive kinase activity by changing the conformation of the sion (ie, quadruple negative carcinomas). intracellular juxtamembrane. Mutation types affecting exon 11 include in-frame deletions, single-nucleotide Gastrointestinal Stromal Tumors substitutions, duplications, and insertions. Deletions are associated

with a poorer prognosis than those with single Overview nucleotide substitutions, whereas duplications in the 3’ Gastrointestinal stromal tumors (GISTs) are biologically region are associated with a better prognosis than other aggressive mesenchymal neoplasms characterized by changes. Approximately 10% of GISTs have mutations KIT mutations in approximately 80% of cases (see also in exon 9 that affect the extracellular domain, whereas Chapter 5). This gene encodes the KIT tyrosine kinase mutations affecting kinase I (exon 13) and kinase II receptor, which is a membrane-associated member of the (exon 17) are less frequent. Exon 9 mutations are more type III tyrosine kinase family. The receptor consists of common among small bowel tumors than those of gas- five loops in its extracellular domain that are connected to tric origin. the cytoplasmic domain via a transmembrane region. The Although KIT mutations are found in most GISTs, cytoplasmic domain contains a juxtamembrane region up to 20% of tumors contain wild-type KIT. Of these, and a split tyrosine kinase domain with two components: nearly 10% harbor mutations in platelet-derived growth an adenosine triphosphate (ATP) binding region (kinase factor receptor alpha polypeptide (PDGFRA), particularly I) and a phosphotransferase region (kinase II). Binding tumors of the stomach and proximal small bowel. This of stem cell factor induces KIT dimerization followed by gene also encodes a tyrosine kinase receptor mapping to phosphorylation of tyrosine residues in the cytoplasmic chromosome 4q11-q12, which is highly homologous to domain, the latter of which activates several signal trans- KIT and shows a parallel distribution of functional muta- duction pathways that drive cell proliferation. tions. Mutations in PDGFRA usually occur in exon 18 and result in conformational changes in the ATP-binding pocket that promote kinase activation. Exon 12 muta- Molecular Alterations tions affecting the juxtamembrane domain and those of Oncogenic KIT mutations impair regulation of recep- exon 14 encoding kinase I are less common. Monosomy, tor activation or alter enzymatic function of tyrosine or partial loss, of chromosome 14q occurs in two-thirds 3 8 8 Neoplastic Gastrointestinal Pathology: An Illustrated Guide of GISTs and 50% show chromosome 22 losses. Losses SELECTED REFERENCES affecting 1p, 9p, and 11p, and gains involving 8q and 17q are associated with aggressive biologic behavior. General GISTs that develop in pediatric patients or in asso- Tanzer M, Liebl M, Quante M. Molecular biomarkers in esopha- ciation with type 1 neurofibromatosis generally lack both geal, gastric, and colorectal adenocarcinoma. Pharmacol Ther. KIT and PDGFRA mutations. Tumors developing in 2013;140(2):133–147. Carney–Stratakis syndrome (ie, GISTs and paraganglio- Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med. 2004;10(8):789–799. mas) frequently show mutations in subunits B, C, or D of SDH, which encode succinate dehydrogenase. Sporadic Barrett Esophagus and Adenocarcinoma GISTs that affect children also contain SDH mutations. They show a predilection for females and usually develop Chandra S, Gorospe EC, Leggett CL, Wang KK. Barrett’s esophagus in 2012: updates in pathogenesis, treatment, and surveillance. Curr in the stomach or omentum. Tumors deficient in succinate Gastroenterol Rep. 2013;15(5):322. dehydrogenase are often resistant to imatinib, but may Gibson MK, Dhaliwal AS, Clemons NJ, et al. Barrett’s esophagus: can- show some response to therapy with sunitinib. cer and molecular biology. Ann NY Acad Sci. 2013;1300:296–314. Kordes S, Cats A, Meijer SL, van Laarhoven HW. Targeted therapy for advanced esophagogastric adenocarcinoma. Crit Rev Oncol Targeted Molecular Therapies Hematol. 2014;90(1):68–76. Lennerz JK, Kwak EL, Ackerman A, et al. MET amplification identifies Imatinib is a tyrosine kinase inhibitor that competes a small and aggressive subgroup of esophagogastric adenocarci- with ATP for its binding site to kinase I and prevents noma with evidence of responsiveness to crizotinib. J Clin Oncol. phosphorylation of cellular substrates. It is highly effec- 2011;29(36):4803–4810. Mayne GC, Hussey DJ, Watson DI. MicroRNAs and esophageal can- tive as a first-line agent against most advanced GISTs cer—implications for pathogenesis and therapy. Curr Pharm Des. and is associated with therapeutic response rates up to 2013;19(7):1211–1226. 90%. Some tumors show diminished response to therapy Norguet E, Dahan L, Seitz JF. Targeting esophageal and gastric over time, reflecting acquisition of additional KIT or cancers with monoclonal antibodies. Curr Top Med Chem. 2012;12(15):1678–1682. PDGFRA mutations that affect imatinib binding to the Okines AF, Gonzalez de Castro D, Cunningham D, et al. Biomarker ATP pocket of the KIT receptor. Sunitinib is a second-line analysis in oesophagogastric cancer: Results from the REAL3 and agent currently approved in patients with imatinib-resis- TransMAGIC trials. Eur J Cancer. 2013;49(9):2116–2125. tant GISTs. This agent can bind to KIT and PDGFRA, Poehlmann A, Kuester D, Malfertheiner P, Guenther T, Roessner A. Inflammation and Barrett’s carcinogenesis. Pathol Res Pract. but also shows affinity for VEGFR. Mutational analy- 2012;208(5):269–280. ses of GISTs are increasingly used to guide patient man- Powell SM, Petersen GM, Krush AJ, et al. Molecular diagnosis of familial agement. Tumors with exon 11 KIT mutations respond adenomatous polyposis. N Engl J Med. 1993;329(27):1982–1987. best to imatinib therapy, followed by those that are KIT/ Rajendra S, Sharma P. Barrett’s Esophagus. Curr Treat Options Gastroenterol. 2014;12(2):169–182. PDGFRA wild-type. GISTs with exon 9 KIT mutations Thompson SK, Sullivan TR, Davies R, Ruszkiewicz AR. Her-2/neu may respond to a higher dose of imatinib or standard gene amplification in esophageal adenocarcinoma and its influence dosing with sunitinib. on survival. Ann Surg Oncol. 2011;18(7):2010–2017. Wainberg ZA, Lin LS, DiCarlo B, et al. Phase II trial of modified FOLFOX6 and erlotinib in patients with metastatic or advanced adenocarcinoma of the oesophagus and gastro-oesophageal junc- SUMMARY AND CONCLUSIONS tion. Brit J Cancer. 2011;105(6):760–765. Winberg H, Lindblad M, Lagergren J, Dahlstrand H. Risk factors Intensive research and technological advances have and chemoprevention in Barrett’s esophagus—an update. Scand J Gastroenterol. 2012;47(4):397–406. enabled investigators to begin unraveling genetic, epi- genetic, and post-translational regulatory mechanisms Gastric Adenocarcinoma that facilitate carcinogenesis in the gastrointestinal tract. Efforts to elucidate cellular signal transduction pathways Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combina- tion with chemotherapy versus chemotherapy alone for treatment continue to unravel relationships between proteins pro- of HER2-positive advanced gastric or gastro-oesophageal junction moting cell survival and proliferation, many of which cancer (ToGA): a phase 3, open-label, randomised controlled trial. represent potential targets for directed medical therapies. Lancet. 2010;376(9742):687–697. Membrane-bound receptor kinases are important candi- Bornschein J, Malfertheiner P. Helicobacter pylori and Gastric Cancer. Dig Dis. 2014;32(3):249–264. dates for inhibitor therapies. Inhibitors of HER-2, EGFR, Camargo MC, Kim WH, Chiaravalli AM, et al. Improved survival of KIT, and other receptor kinases are effective in the treat- gastric cancer with tumour Epstein-Barr virus positivity: an inter- ment of patients with specific gastrointestinal malignan- national pooled analysis. Gut. 2014;63(2):236–243. cies. Future management strategies will likely include the Chen S, Duan G, Zhang R, Fan Q. Helicobacter pylori cytotoxin-asso- ciated gene A protein upregulates alpha-enolase expression via Src/ use of these agents, as well as other therapies aimed at MEK/ERK pathway: Implication for progression of gastric cancer. specific molecular alterations. Int J Oncol. 2014;45(2):764–770. 14 Applications of Molecular Pathology 3 8 9 Hirahashi M, Koga Y, Kumagai R, Aishima S, Taguchi K, Oda Y. Induced Ligtenberg MJ, Kuiper RP, Chan TL, et al. Heritable somatic meth- nitric oxide synthetase and peroxiredoxin expression in intramuco- ylation and inactivation of MSH2 in families with Lynch syn- sal poorly differentiated gastric cancer of young patients. Path Int. drome due to deletion of the 3’ exons of TACSTD1. Nat Genet. 2014;64(4):155–163. 2009;41(1):112–117. Martinez-Lopez JL, Torres J, Camorlinga-Ponce M, Mantilla A, Leal Samowitz WS. The CpG island methylator phenotype in colorectal YA, Fuentes-Panana EM. Evidence of Epstein-Barr virus asso- cancer. The J Mol Diag. 2007;9(3):281–283. ciation with gastric cancer and non-atrophic gastritis. Viruses. Shen L, Toyota M, Kondo Y, et al. Integrated genetic and epigenetic 2014;6(1):301–318. analysis identifies three different subclasses of colon cancer. Proc Nishikawa J, Yanai H, Hirano A, et al. High prevalence of Epstein-Barr Natl Acad Sci U S A. 2007;104(47):18654–18659. virus in gastric remnant carcinoma after Billroth-II reconstruc- Spirio LN, Samowitz W, Robertson J, et al. Alleles of APC modulate the tion. Scand J Gastroenterol. 2002;37(7):825–829. frequency and classes of mutations that lead to colon polyps. Nat Okada T, Nakamura M, Nishikawa J, et al. Identification of genes spe- Genet. 1998;20(4):385–388. cifically methylated in Epstein-Barr virus-associated gastric carci- nomas. Cancer Sci. 2013;104(10):1309–1314. Ruschoff J, Hanna W, Bilous M, et al. HER2 testing in gastric cancer: a Gastrointestinal Stromal Tumors practical approach. Mod Pathol. 2012;25(5):637–650. Barnett CM, Corless CL, Heinrich MC. Gastrointestinal stromal Shimizu T, Marusawa H, Matsumoto Y, et al. Accumulation of Somatic tumors: molecular markers and genetic subtypes. Hematol Oncol Mutations in TP53 in Gastric Epithelium with Helicobacter pylori Clin North Am. 2013;27(5):871–888. infection. Gastroenterology. 2014;147(2):407–17. Beadling C, Patterson J, Justusson E, et al. Gene expression of the Watari J, Chen N, Amenta PS, et al. associated chronic gastri- IGF pathway family distinguishes subsets of gastrointestinal tis, clinical syndromes, precancerous lesions, and pathogen- stromal tumors wild type for KIT and PDGFRA. Cancer Med. esis of gastric cancer development. World J Gastroenterol. 2013;2(1):21–31. 2014;20(18):5461–5473. Demetri GD, Benjamin R, Blanke CD, et al. NCCN Task Force report: optimal management of patients with gastrointestinal stromal Colorectal Cancer tumor (GIST)—expansion and update of NCCN clinical practice guidelines. J Natl Compr Canc Netw. 2004;2 Suppl 1:S-1–S-26. Albuquerque C, Breukel C, van der Luijt R, et al. The ‘just-right’ signal- Demetri GD, Benjamin RS, Blanke CD, et al. NCCN Task Force report: ing model: APC somatic mutations are selected based on a specific management of patients with gastrointestinal stromal tumor level of activation of the beta-catenin signaling cascade. Hum Mol (GIST)—update of the NCCN clinical practice guidelines. J Natl Genet. 2002;11(13):1549–1560. Compr Canc Netw. 2007;5 Suppl 2:S1–S29. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Espinosa I, Lee CH, Kim MK, et al. A novel monoclonal antibody Gastroenterology. 2010;138(6):2073–2087 e3. against DOG1 is a sensitive and specific marker for gastrointesti- Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer nal stromal tumors. Am J Surg Pathol. 2008;32(2):210–218. Institute Workshop on Microsatellite Instability for cancer detec- Heinrich MC, Maki RG, Corless CL, et al. Primary and secondary tion and familial predisposition: development of international cri- kinase genotypes correlate with the biological and clinical activity teria for the determination of microsatellite instability in colorectal of sunitinib in imatinib-resistant gastrointestinal stromal tumor. cancer. Cancer Res. 1998;58(22):5248–5257. J Clin Oncol. 2008;26(33):5352–5359. Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treat- Janeway KA, Liegl B, Harlow A, et al. Pediatric KIT wild-type and ment and RAS mutations in colorectal cancer. N Engl J Med. platelet-derived growth factor receptor alpha-wild-type gastroin- 2013;369(11):1023–1034. testinal stromal tumors share KIT activation but not mechanisms Durno CA, Holter S, Sherman PM, Gallinger S. The gastrointestinal of genetic progression with adult gastrointestinal stromal tumors. phenotype of germline biallelic mismatch repair gene mutations. Canc Res. 2007;67(19):9084–9088. Am J Gastroenterol. 2010;105(11):2449–2456. Lasota J, Corless CL, Heinrich MC, et al. Clinicopathologic profile of Kane MF, Loda M, Gaida GM, et al. Methylation of the hMLH1 pro- gastrointestinal stromal tumors (GISTs) with primary KIT exon 13 moter correlates with lack of expression of hMLH1 in sporadic or exon 17 mutations: a multicenter study on 54 cases. Mod Pathol. colon tumors and mismatch repair-defective human tumor cell 2008;21(4):476–484. lines. Cancer Res. 1997;57(5):808–811. Mason EF, Hornick JL. Succinate dehydrogenase deficiency is asso- Lamlum H, Ilyas M, Rowan A, et al. The type of somatic mutation at ciated with decreased 5-hydroxymethylcytosine production in APC in familial adenomatous polyposis is determined by the site of gastrointestinal stromal tumors: implications for mechanisms of the germline mutation: a new facet to Knudson’s ‘two-hit’ hypoth- tumorigenesis. Mod Pathol. 2013;26(11):1492–1497. esis. Nat Med. 1999;5(9):1071–1075. Pantaleo MA, Astolfi A, Urbini M, et al. Analysis of all subunits, Lefevre JH, Colas C, Coulet F, et al. MYH biallelic mutation can inac- SDHA, SDHB, SDHC, SDHD, of the succinate dehydrogenase tivate the two genetic pathways of colorectal cancer by APC or complex in KIT/PDGFRA wild-type GIST. Eur J Hum Genet. MLH1 transversions. Fam Cancer. 2010;9(4):589–594. 2014;22(1):32–39. Liao X, Lochhead P, Nishihara R, et al. Aspirin use, tumor PIK3CA Vadakara J, von Mehren M. Gastrointestinal stromal tumors: manage- mutation, and colorectal-cancer survival. N Engl J Med. ment of metastatic disease and emerging therapies. Hematol Oncol 2012;367(17):1596–1606. Clin North Am. 2013;27(5):905–920. Index Adenocarcinoma Vs. signet ring cell change, 196 Hereditary cancer syndromes and, Anus, 324 Vs. reactive changes, 197 222 Clinical/macroscopic features, 324 Early gastric

cancer, 190 Adenomatous polyposis syndromes, 121 Epidemiology/pathogenesis, 324 Epidemiology, 188 Adnexal tumors, anus, 330 Histologic features, 324 Genetic syndromes and, 193 Anal tags/fibroepithelial polyps, 317 Immunohistochemistry, 325 Hereditary diffuse gastric cancer, APC gene, 384 Vs. rectal adenocarcinoma, 325 193, 383 Appendiceal mucinous neoplasms, 250 Appendiceal, 254, 257 CDH1, 193 Adenoma/cystadenoma, 251 Colorectal, 283 Grading/staging, 195 Differential diagnosis, 257 Clinical features, 284 HER2 and, 195, 360, 383 Adenocarcinoma vs. LAMN, 257 Differential diagnosis, 289 Immunohistochemistry, 195 Adenoma vs. LAMN, 257 Vs. endometriosis/ Lynch syndrome and, 194 LAMN vs. diverticulum, 257 endosalpingiosis, 289 Management/prognosis, 195 LAMN vs. hyperplasia, 257 Vs. misplaced epithelium, 289 MSI-H and, 194 Immunohistochemistry, 257 Disease associations, 285 Molecular features, 195, 382 Low grade appendiceal mucinous Epidemiology, 283 Pathologic features, 190 neoplasms (LAMN), 252 Grading and staging, 291 Variants, 190 Molecular features, 257 Challenges in, 292 Small bowel Mucinous adenocarcinoma, 254 Immunohistochemistry, 284 Clinical features, 224 Pseudomyxoma peritonei, 254 Lymph node assessment, 295 Vs. colorectal adenocarcinoma, Appendiceal nonmucinous neoplasms, Vs. tumor deposit, 297 225 257 Lymphovascular invasion, 298 Differential diagnosis, 226 Molecular features, 300, 384 Vs. adenocarcinoma with high Barrett esophagus, 155 Neoadjuvant chemoradiation and, grade dysplasia, 226 Dysplasia and, 157 299 Vs. ectopic pancreas, 227 Crypt dysplasia, 159 Pathologic features, 284 Vs. endometriosis, 227 Differential diagnosis, 162 Presenting in adenoma, 289 Vs. pancreatic adenocarcinoma, Vs. reactive changes, 162 Total mesorectal excision and, 295 227 Foveolar dysplasia, 158 Tumor budding, 298 Disease associations, 224 Grading, 157 Variants and subtypes, 284 Epidemiology, 222 Molecular features, 380 Esophageal, 155 Grading and staging, 228 p53 and, 352 Clinical/endoscopic features, 156 Immunohistochemistry, 225 Treatment and surveillance, 159 Differential diagnosis, 162 Molecular features, 225 Basal cell carcinoma, anal, 329 Intramucosal carcinoma vs. high Pathologic features, 225 Vs. basaloid squamous cell carcinoma, grade dysplasia, 163 Adenoma, intestinal type 329, 371 Epidemiology/pathogenesis, 155 Appendiceal, 257 Biopsy specimens, general approach to, Grading/staging, 166 Colorectal, 271 15 HER2 and, 162 “Advanced” adenoma, 273 BRAF V600E immunohistochemistry, Histologic variants, 163 Architectural patterns, 271 365 Immunohistochemistry, 161 Carcinoma in situ and, 273 Brunner gland hyperplasia, 217 Poorly differentiated carcinoma Differential diagnosis, 274 Burkitt lymphoma, 81 and, 357 Vs. IBD-associated dysplasia, Small bowel, 241 Management/prognosis, 156 274 Vs. “double hit” lymphoma, 243 Molecular features, 161 Vs. reactive changes, 274 Gastric High grade dysplasia and, 271 Advanced gastric carcinoma, 190 Metaplasia and, 271 Carcinoma in situ Clinical features, 189 Treatment and surveillance, 273 Alternative classifications, 5 Differential diagnosis, 196 Small bowel, 221 Terminology, 5 3 91 3 9 2 Index Clear cell sarcoma, gastrointestinal, 236 Gangliocytic paraganglioma, 114, 229 Granular cell tumor, 104 Colitis cystica profunda, 266 Ganglioneuroma, 104 Anus, 330 Colorectal neoplasia, classification of, Colorectal, 303 Colorectal, 303 265 Ganglioneuromatosis, 104 Esophageal, 167 CpG island methylator phenotype, 386 Gastric neoplasia, classification of, 174 Cronkhite–Canada syndrome Gastrointestinal neoplasia Hamartomatous polyposis syndromes, Colorectal, 270 Grading, 7 127 Gastric, 181 Eastern vs. western classification, 6 Colorectum, 269 Prognostic and predictive markers, 13 Small bowel, 218 Desmoid tumor, 108 Reporting, 12 Hemangioma, small bowel, 235 Small bowel, 234 Risk factors for, 2 Hematolymphoid neoplasms Diagnosis and reporting, introduction Screening and surveillance for, 14 Colorectal, 303 to, 1 Staging, 10 Gastric, 205 Diffuse large B cell lymphoma, 79, 209 Terminology, 1 MALT lymphoma, 206 EBV and, 209 Gastrointestinal stromal tumor (GIST), Vs. H. pylori gastritis, 208 Differential diagnosis of, 211 95 Vs. mantle cell lymphoma, 208 Vs. MALT lymphoma, 211 Dedifferentiated, 98 Vs. plasmacytic neoplasms, 208 Vs. mantle cell lymphoma, 211 Differential diagnosis, 101 General approach to, 75, 78, 82 Immunohistochemistry, 210 Esophageal, 169 Illustrative examples, 83 Molecular features, 210 Gastric, 202 Immunohistochemistry, 339 Pathologic features, 209 Carney triad and, 203 Small bowel, 238 Diverticular polyps (polypoid prolapsing Carney–Stratakis syndrome and, Hemorrhoids, 316 mucosal folds), 265 203 HER2 Dysplasia (see also Glandular and Differential diagnosis, 203 Esophageal neoplasia and, 162, 381 Squamous dysplasia) Grading and prognosis, 203 Gastric neoplasia and, 195, 383 Terminology, 3 Immunohistochemistry, 203 Immunohistochemistry, 360 Introduction to grading, 3 DOG1 and, 203 Hereditary cancer syndromes, 121 KIT and, 203 Illustrative examples, 135 Molecular features, 203 Endometriosis/endosalpingiosis, 224 Heterotopic gastric mucosa BRAF V600E mutations, 203 Vs. neoplasia, 24, 227, 289 Small bowel, 217 PDGFRA and, 203 Enteropathy-associated T-cell lymphoma Heterotopic mucosa, esophagus, 144 Pathologic features, 202 (EATL), 238 Heterotopic pancreas, 24 SDH-deficient, 202 Celiac disease and, 238 Gastric, 175 Grading and prognosis, 100 EPCAM immunohistochemistry, 365 Small bowel, 217 Immunohistochemistry, 99, 342 Epidermoid metaplasia, esophagus, 147 Vs. neoplasia, 24, 227 Molecular features, 100, 387, 388 Epithelial neoplasms Hyperplastic polyp Pathologic features, 96 Classification, 19 Colorectal, 276 SDH deficient, 99, 388 Differential diagnosis, 27 Vs. sessile serrated adenoma, 282 Small bowel, 232 Epidemiology, general, 19 Gastric, 173 Syndromic involvement, 101 General approach to diagnosis, 27 Differential diagnosis, 178 Treatment, 101 Illustrative examples, 33 Vs. dysplasia, 178 Giant fibrovascular polyp, esophagus, Immunohistochemistry in, 30, 331 Dysplasia in, 174 169 Esophageal neoplasia, classification, 142 Glandular dysplasia (see also Barrett Extranodal NK/T cell lymphoma, nasal esophagus) Immunohistochemistry, general type, 239 Gastric approach to Differential diagnosis, 185 CK7/CK20 expression patterns, 334 Familial adenomatous polyposis, 121 Vs. reactive changes, 185 Epithelial neoplasms and, 331 Clinical features, 122 Eastern vs. western classification, Hematolymphoid neoplasms and, 339 Differential diagnosis, 123 183 Mesenchymal neoplasms and, 342 Molecular features, 121, 384 Foveolar dysplasia, 183 Neuroendocrine neoplasms and, 334 Pathologic features, 122 Gross appearance, 183 Primary vs. metastatic neoplasia and, Follicular center cell lymphoma, 80 Intestinal type, 183 332 Small bowel, 239 Morphologic classification, 183 Inflammatory cap polyposis, 265 Foveolar hyperplasia, polypoid, 173 Risk factors, 183 Inflammatory cloacogenic polyp, 266 Fundic gland polyp, 178 Glomus tumor, 109, 204 Inflammatory fibroid polyp, 112 Familial adenomatous polyposis and, Glycogenic acanthosis, 141 Gastric, 204 178 Goblet cell carcinoid tumor, 259 Small bowel, 234 Index 3 9 3 Inflammatory myofibroblastic tumor, MALT lymphoma, gastric (see also Differential diagnosis, 124 112 Hematolymphoid neoplasia, Molecular features, 124 Inflammatory myoglandular polyp, 266 gastric), 77, 79, 206 Pathologic features, 124 Inflammatory polyp Mammary analog tumors, anus, 330 Colorectal, 268 Mantle cell lymphoma, 80 Esophageal, 142 Colorectal, 303 Neuroendocrine carcinoma (NEC) Gastric, 173 Melanoma Colorectal, 301 Vs. dysplasia, 178 Anus, 328 Epidemiology, 44 Small bowel, 217 Epidemiology, 328 General features, 42 Intramucosal carcinoma Pathologic features, 328 Neuroendocrine neoplasia Alternative classifications, 5 Esophagus, 170 Classification of, 44 Terminology, 5 Mesenchymal neoplasia Clinical significance of, 55 Approach to, 87, 89 Diagnosis of, 59, 334 Juvenile polyposis syndrome, 130 Classification by location, 88 Differential diagnosis of, 56 Clinical features, 130 Clinical features, 87 General approach to, 39 Colorectal, 269 Gross evaluation, 88 Grading, 59 Differential diagnosis, 131 Histologic features, 88 Historical perspective, 39 Gastric, 179 Illustrative examples, 115 Hormone production in, 41 Molecular features, 130 Immunohistochemistry, 90, 342 Illustrative examples, 66 Pathologic features, 130 CD34, 94 Metastatic, 62 Small bowel, 218 Desmin, 93 Reporting, 59, 65 DOG1, 90 Neuroendocrine tumors (NET) Keratins, 94 Appendiceal, 52, 249 Kaposi sarcoma, small bowel, 235 KIT, 90, 351 Behavior/management, 250 KIT immunohistochemistry, 90, 351 S100, 92 Enterochromaffin cell tumors, 249 KIT mutations, 387 SMA, 93 Enteroglucagon cell tumors, 249 Carney–Stratakis syndrome and, Intraoperative evaluation and, 88 Reporting, 250 388 Molecular features, 96 Colorectal, 52, 301 NF1 and, 388 Metastatic tumors Epidemiology and clinical features, Involving colon, 309 301 Langerhans cell histiocytosis, colorectal, Colon vs. breast primary, 309 Staging, 301 307 Colon vs. Müllerian primary, 309 Vs. colorectal carcinoma, 301 Leiomyoma, 102 Involving esophagus, 170 Vs. prostate carcinoma, 301 Colorectal, 302 Involving small bowel, 244 Epidemiology, 42 Esophagus, 169 Involving stomach, 197 Esophageal, 47, 163 Leiomyomatosis, 169 Gastric vs. breast primary, 197, Gastric, 47, 198 Small bowel, 232 359 Atrophic gastritis and, 199 Leiomyomatosis peritonealis MicroRNAs, 380 Clinical features, 199 disseminata, 104 Microsatellite instability, 384 Differential diagnosis, 201 Leiomyosarcoma, 114 Lynch syndrome and, 384 Epidemiology, 198 Esophagus, 169 Sporadic tumors, 385 Grading and staging, 201 Small bowel, 232 Mismatch Repair (MMR) protein Pathologic features, 199 Lipomatous hypertrophy of ileocecal immunohistochemistry, 125, Zollinger–Ellison syndrome and, valve, 234 362 199 Lymphangioma, small bowel, 235 Pitfalls, 365 General features, 41 Lymphoid antigens, 75, 339 Polyps and, 367 Immunohistochemistry by site, 65, Lymphoid polyps, rectal, 308 Mixed adenoneuroendocrine carcinoma 334 Lynch syndrome, 124 (MANEC), 46 Small bowel, 48, 51, 228 Clinical features, 125 Colorectal, 301 Differential diagnosis, 229 Differential diagnosis, 127 Gastric, 199 Vs. adenocarcinoma, 229 MMR immunohistochemistry, 125, Molecular testing in gastrointestinal Duodenal, 48 362 neoplasia, 379 Epidemiology and clinical features, Molecular features, 124, 384 Mucosal prolapse polyps 228 Pathologic features, 125 Colorectal, 265 Grading/Staging, 230 Vs. neoplasia, 25, 267 Jejunoileal, 51 Malignancy Gastric, 175 Molecular features, 229 Diagnosis of, 20 MUTYH-associated polyposis, 124 Pathologic features, 229 Primary vs. metastatic, 21, 332 Clinical features, 124 Neurofibroma, 108 3 9 4 Index Oncogenes, 379 Differential diagnosis, 188 Variants, 322 Oxyntic gland polyp/adenoma, 188 MUC5AC and, 188 Esophagus, 145 Clinical/endoscopic features, 145 p16 immunohistochemistry, 318, 320, Differential diagnosis, 151 Resection specimens, general approach 368 Epidemiology/pathogenesis, 145 to, 17 Paget disease, perianal, 326 Immunohistochemistry, 149 Clinical/macroscopic features, 326 Management/prognosis, 146 Differential diagnosis, 327 Schwann cell hamartoma, mucosal, 104 Molecular studies, 149 Vs. anal adenocarcinoma, 327 Colorectal, 303 Pathologic features, 146 Vs. melanoma, 327 Schwannoma, 104 Variants, 148 Vs. squamous cell carcinoma, 328 Colorectal, 303 Squamous dysplasia Etiology, 326 Gastric, 204 Anus, 317 Immunohistochemistry, 326, 370 SDH mutations, 388 Clinical/macroscopic features, 319 Primary vs. secondary, 326 Serrated lesions of the appendix, 259 Condyloma and, 320 PDGFRA mutations, 387 Dysplasia and, 259 Differential diagnosis, 320 PEComa, 112 Serrated polyps, colorectal, 276 Vs. glycogenated nuclei, 320 Perineurioma, 107 Perineurioma-like changes in, 279 Vs. inflammatory/reactive Peutz–Jegher syndrome, 127 Treatment/surveillance, 282 transition zone, 320 Clinical features, 128 Sessile serrated adenoma/polyp, 276 p16 immunohistchemistry and, Colorectal, 270 Differential diagnosis, 282 320 Differential diagnosis, 129 Vs. hyperplastic polyp, 282 Epidemiology, 317 Gastric, 179 Vs. inflammatory or prolapse polyp, Histologic features, 319 Molecular features, 127 283 HPV and, 318 Pathologic features, 128 Vs. traditional serrated adenoma, HSIL, 319 Small bowel, 218 283 LSIL, 319 Peptic duodenitis, vs. dysplasia, 217 Dysplasia and, 279 p16, 318, 320 Plexiform myxofibroma, 112, 204 Ki-67, 279 Terminology, 318 Pseudo-signet ring cells, 27, 196 Molecular alterations, 282 Esophagus, 145 PTEN hamartomatous polyposis Sessile serrated polyposis syndrome, 281 Vs. reactive changes, 151 syndrome, 132 Small bowel neoplasia Squamous papilloma, 141 Clinical features, 133 Classification, 217 Endophytic, 142 Colorectal, 270 Clinical features, 218 Exophytic, 142 Differential diagnosis, 133 Squamous cell carcinoma Spiked, 142 Molecular features, 132 Anus, 321 Systemic mastocytosis, colorectal, 306 Pathologic features, 133 Clinical/macroscopic features, 322 Pyloric gland adenoma Epidemiology/pathogenesis, 321 T cell lymphomas, 81 Esophagus, 170 Histologic features, 322 Tumor suppressor genes, 379 Gastric, 187 Staging, 323 Tyrosine kinase inhibitors, 381, 388

ANTICANCER AGENTS from NATURAL PRODUCTS Edited by Gordon M. Cragg David G.I. Kingston David J. Newman Boca Raton London New York Singapore A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc. Copyright © 2005 CRC Press, LLC Published in 2005 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2005 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-1863-7 (Hardcover) International Standard Book Number-13: 978-0-8493-1863-4 (Hardcover) Library of Congress Card Number 2004065568 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Kingston, David. Anticancer agents from natural products / David Kingston, Gordon Cragg, David Newman. p. cm. ISBN 0-8493-1863-7 1. Antineoplastic agents. 2. Pharmacognosy. 3. Natural products. I. Cragg, Gordon M. L. II. Newman, David J. III. Title. RS431.A64K545 2005 616.99'4061--dc22 2004065568 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group and the CRC Press Web site at is the Academic Division of T&F Informa plc. http://www.crcpress.com Copyright © 2005 CRC Press, LLC Preface Natural products have made an enormous contribution to cancer chemotherapy, and over half of the current anticancer agents in clinical use are natural products or are derived from natural products. In spite of this fact, no book published in recent years has brought together the disparate information on anticancer natural products that is currently scattered throughout the chemical, biological, and medical literature. The most recent book to do this was Anticancer Agents Based on Natural Product Models, edited by John Cassady and John Douros, but this text was published in 1980 and is now sadly out of date. The present book covers the current clinically used anticancer agents that are either natural products or are clearly derived from natural product leads. In addition, a number of drug candidates that are in clinical development are also covered, albeit more briefly, as many of these will be clinically used drugs in the future. It is expected that this volume will appeal to several classes of reader. It will be of interest to natural products chemists, medicinal chemists, and pharmacognosists as an important reference work in their area of interest. It will appeal to synthetic organic chemists as a source of information on challenging synthetic targets. It will also appeal to oncologists as a source of background information on the drugs they use, although it is not intended as a primary clinical text. Some of the key features of the book include up-to-date coverage of a field that is scattered among many different journals and review articles, inclusion of information on drugs in clinical development, and authorship by leading scientists on each drug; in some cases the drug developer or a close associate is the chapter author. These features should give the book real value for scientists looking for information on the next generation of anticancer drugs. The editors express their appreciation to the staff of Taylor & Francis for their excellent help, especially Randi Cohen, Erika Dery, Lindsey Hofmeister, and Jay Margolis. The volume is dedicated to the memory of those scientists who have blazed the trail to successful natural products-based anticancer drug development, including Drs. John Douros, John Faulkner, Jonathan Hartwell, Morris Kupchan, Paul Scheuer, Matthew Suffness, and Monroe Wall. May their tribe increase! Gordon M. Cragg, David G. I. Kingston, and David J. Newman Frederick, MD, and Blacksburg, VA Copyright © 2005 CRC Press, LLC Editors Gordon M. Cragg was born in Cape Town, South Africa, and obtained his undergraduate training in chemistry at Rhodes University before proceeding to Oxford University, where he obtained his D. Phil. in organic chemistry in 1963. After 2 years of postdoctoral research in natural products chemistry at the University of California, Los Angeles, he returned to South Africa to join the Council for Scientific and Industrial Research. In 1966, he was appointed to the staff of the Department of Chemistry at the University of South Africa and transferred to the University of Cape Town in 1972. In 1979, he returned to the United States to join the Cancer Research Institute at Arizona State University, working with Professor G. Robert Pettit on the isolation of potential anticancer agents from plant and marine invertebrate sources. In 1985, he moved to the National Cancer Institute in Bethesda, Maryland, and was appointed Chief of the Natural Products Branch in 1989. His major interests lie in the discovery of novel natural product agents for the treatment of cancer and AIDS. He has been awarded National Institutes of Health Merit Awards for his contributions to the development of the drug, taxol (1991), leadership in establishing international collaborative research in biodiversity and natural products drug discovery (2004), and contributions to developing and teaching NIH technology transfer courses (2004). In 1998–1999 he served as President of the American Society of Pharmacognosy, and was elected to honorary membership of the society in 2003. He has established collaborations between the National Cancer Institute and organizations in many countries promoting drug discovery from their natural resources. He has given over 100 invited talks at conferences in many countries and has published more than 140 papers related to these interests. David G. I. Kingston was born in London, England, and obtained both his undergraduate and graduate training in chemistry at Cambridge University. His graduate supervisors were Lord Todd and Dr. D. W. Cameron, and his Ph.D. research was on the chemistry of the aphid pigments; he completed his Ph.D. degree in 1963. He then did 3 years of postdoctoral research; one in the Division of Biochemistry at MIT under Professor J. M. Buchanan and two back at Cambridge, where he was a Research Fellow of Queens College and worked with Lord Todd, Franz Sondhe- imer, and Dudley Williams. He moved to the State University of New York at Albany in 1966 and then to Virginia Polytechnic Institute and State University in 1971, where he currently holds the rank of University Distinguished Professor. He served as President of the American Society of Pharmacognosy in 1988–1989. His research interests are on the isolation and structure elucidation of novel natural products, especially those with anticancer activity, and on the chemistry and mechanism of action of tubulin-binding natural anticancer agents such as taxol, epothilone, and discodermolide, and he currently serves as the principal investigator of the Madagascar Interna- tional Cooperative Biodiversity Group. He received the Research Achievement Award of the American Society of Pharmacognosy in 1999 and was named Virginia Scientist of the Year in 2002. He has published over 280 papers and holds 14 patents. He is also an Elder in his church, the Blacksburg Christian Fellowship. David J. Newman was born in Grays, Essex. Initially he trained as a chemical analyst (Grad. RIC), followed by his being awarded an M.Sc. in organic chemistry (University of Liverpool), and then after time in the U.K. chemical industry, he obtained a D. Phil. in microbial chemistry from the University of Sussex in 1968. Following two years of postdoctoral studies on the structure of electron transport proteins at the University of Georgia, he worked for Smith Kline and French in Copyright © 2005 CRC Press, LLC Philadelphia, Pennsylvania, as a biological chemist predominantly in the area of antibiotic discovery. During this time, he obtained an M.S. in information sciences in 1977 from Drexel University, Philadelphia. He has worked for a number of United States-based pharmaceutical companies in natural products-based discovery programs in antiinfective and cancer treatments and joined the Natural Products Branch of the National Cancer Institute in 1991. He is responsible for the marine and microbial collection programs of the National Cancer Institute and, in concert with Gordon Cragg, for the National Cancer Institute’s Open and Active Repository programs. In 2003 he was awarded the National Institutes of Health Merit Award for his contributions to the development of potential anticancer agents from marine and microbial sources. His scientific interests are in the discovery and history of novel natural products as drug leads in the antiinfective and cancer areas and in the application of information technologies to drug discovery. In conjunction with Gordon Cragg, he has established collaborations between the National Cancer Institute and organizations in many countries promoting drug discovery from their natural resources. He has published over 60 papers, presented over 60 abstracts, holds 17 patents that are related to these interests, is both a UK Chartered Chemist and a UK Chartered Biologist, and is also an adjunct full professor at the Center of Marine Biotechnology, University of Maryland. Copyright © 2005 CRC Press, LLC Contributors Dr. Rima Al-awar Dr. Gordon M. Cragg Eli Lilly and Company Natural Products Branch Lilly Corporate Center Developmental Therapeutics Program Discovery Chemistry Research Division of Cancer Treatment and Diagnosis and Technology National Cancer Institute Drop Code 2810 NCI-Frederick Indianapolis, IN 46285 Fairview Center, Room 206 AL-AWAR_RIMA_S@LILLY.COM P.O. Box B Frederick, MD 21702-1201 craggg@mail.nih.gov Professor Raymond J. Andersen Dept of Chemistry Dr. Carmen Cuevas University of British Columbia PharmaMar Vancouver, BC Av. de los Reyes, 1, P.I. La Mina Canada V6T 1Z1 28770 Colmenar Viejo randersn@interchange.ubc.ca Madrid, Spain ccuevas@pharmamar.com Dr. Klaus Edvardsen Dr. Federico Arcamone Department of Cell and Molecular Biology Via Quattro Novembre 26 Section for Tumor Immunology Nerviano University of Lund 20014 I12, 221 84 Milano, Italy Lund, Sweden farcamone@virgilio.it Dr. Jacques Fahy Division de Chimie Médicinale 5 Dr. Kathryn A. Bixby Centre de Recherches Pierre F. Department of Bacteriology 17, avenue Jean Moulin University of Wisconsin-Madison 81106 CASTRES, France 777 Highland Avenue Jacques.fahy@pierre-fabre.com Madison, WI 53705-2222 Dr. Glynn T. Faircloth PharmaMar USA, Inc. 320 Putnam Avenue Dr. Don Borders Cambridge, MA 02139 13 Heatherhill Lane gfaircloth@pharmamarusa.com Suffern, NY 10901 biosource@prodigy.net Dr. Erik Flahive Pfizer Global Research and Development - La Jolla Labs Dr. David J. Chaplin 10578 Science Center Drive Oxigene Inc. San Diego, CA 92109 230 Third Avenue Erik.flahive@pfizer.com Waltham, MA 02451 eflahive@earthlink.net Copyright © 2005 CRC Press, LLC Professor Heinz G. Floss Professor Hideji Itokawa University of Washington Department of Medicinal Chemistry, School of Department of Chemistry Pharmacy Campus Box 351700 Beard Hall CB7360 Seattle, WA 98195-1700 University of North Carolina floss@chem.washington.edu Chapel Hill, NC 27599-7360 Dr. (Mrs) Françoise Guéritte Dr. Christopher Jelinek I.C.S.N./C.N.R.S. Department of Chemistry and Biochemistry Avenue de la Terrasse P.O. Box 97348 91198 Gif-sur-Yvette, France Baylor University Francoise.Gueritte@icsn.cnrs-gif.fr Waco, Texas 76798-7348 Dr. Sarath P. Gunasekera David G. I. Kingston Harbor Branch Oceanographic Institution Department of Chemistry, M/C 0212 5600 US 1 North Virginia Polytechnic Institute and State Fort Pierce, FL 34946 University sgunaseker@hboi.edu Blacksburg, VA 24061 DKingston@vt.edu Dr. Philip R. Hamann Chemical and Screening Sciences Professor Yoshita Kishi Wyeth Research Laboratories Dept. of Chemistry 401 N. Middletown Road Harvard University Pearl River, NY 10965 Cambridge, MA 02138 HAMANNP@wyeth.com Kishi@chemistry.harvard.edu Professor Sidney M. Hecht Dr. Rohtash Kumar Department of Chemistry Department of Chemistry University of Virginia University of Alberta McCormick Road, PO Box 400319 Edmonton Charlottesville, VA 22904-4319 Alberta T6G

2G2 sidhecht@virginia.edu Canada Dr. Rubén Henríquez Professor Helmut Lackner PharmaMar Institut für Av. de los Reyes, 1, P.I. La Mina Universität Göttingen 28770 Colmenar Viejo Tammannstraße 2 Madrid, Spain D-37077 rhenriquez@pharmamar.com Göttingen, Germany hlackne@gwdg.de Dr. Gerhard Höfle Bereich Naturstoffe Profesor K. H. Lee GBF – Gesellschaft für Biotechnologische Department of Medicinal Chemistry, School of Forschung Pharmacy Mascheroder Weg 1 Beard Hall CB7360 D-38124 University of North Carolina Braunschweig, Germany Chapel Hill, NC 27599-7360 gho@gbf.de khlee@unc.edu Copyright © 2005 CRC Press, LLC Dr. Bruce A. Littlefield Dr. Michelle Prudhomme Eisai Research Institute Université Blaise Pascal One Corporate Drive Laboratoire de Synthèse et Etude de Systèmes Andover, MA 01810 à Intérêt Biologique bruce_littlefield@eri.eisai.com UMR 6504 du CNRS URA 485 du CNRS Professor J. W. Lown 63177 Department of Chemistry Aubière, France University of Alberta mprud@chimtp.univ-bpclermont.fr Edmonton Alberta T6G 2G2 Dr. Nicholas J. Rahier Canada Department of Chemistry Lynne.lechelt@ualberta.ca University of Virginia McCormick Road, PO Box 400319 Dr. Anthony Mauger Charlottesville, VA 22904-4319 10206 Frederick Ave Kensington, MD 20891-3304 Professor Hans Reichenbach maugerai@yahoo.com Bereich Naturstoffe Gesellschaft für Biotechnologische Forschung Dr. David J. Newman Mascheroder Weg 1 Natural Products Branch D-38124 Developmental Therapeutics Program Braunschweig, Germany Division of Cancer Treatment and Diagnosis hre@gbf.de National Cancer Institute NCI-Frederick Dr. William Remers Fairview Center, Room 206 AmpliMed Corporation P.O. Box B 4280 N. Campbell Avenue Frederick, MD 21702-1201 Tucson, AZ 85718 newmand@mail.nih.gov remers@pharmacy.arizona.edu Professor George R. Pettit Professor Michel Roberge Cancer Research Institute Department of Biochemistry and Molecular Arizona State University, Main Campus Biology P.O. Box 872404 University of British Columbia Tempe, AZ 85287-2404 Vancouver, BC Canada V6T 1Z1 Professor Kevin G. Pinney michelr@interchange.ubc.ca Department of Chemistry and Biochemistry P.O. Box 97348 Professor Ben Shen Baylor University Division of Pharmaceutical Sciences Waco, TX 76798-7348 School of Pharmacy Kevin_Pinney@baylor.edu University of Wisconsin-Madison 777 Highland Avenue Madison, WI 53705-2222 bshen@pharmacy.wisc.edu Copyright © 2005 CRC Press, LLC Dr. Chuan Sih Dr. Janis Upeslacis Eli Lilly and Company Chemical and Screening Sciences Lilly Corporate Center Wyeth Research Laboratories Discovery Chemistry Research and Technology 401 N. Middletown Road Indianapolis, IN 46285 Pearl River, NY 10965 SHIH-CHUAN@LILLY.COM UPESLAJ@wyeth.com Dr. Kenneth Snader Dr. Amy E. Wright 1346 34th Avenue Harbor Branch Oceanographic Institution Vero Beach, FL 32960 5600 US 1 North snaderk@yahoo.com Fort Pierce, FL 34946 wright@hboi.edu Dr. Jay Srirangam Chemical Research & Development Ms. Zhiyan Xiao Pfizer Global R&D - La Jolla Labs Department of Medicinal Chemistry, School of 10578 Science Center Drive Pharmacy San Diego, CA 92109 Beard Hall CB7360 Jay.srirangam@pfizer.com University of North Carolina Chapel Hill, NC 27599-7360 Dr. Craig J. Thomas Department of Chemistry Dr. Melvin Yu University of Virginia Eisai Research Institute McCormick Road, PO Box 400319 4 Corporate Drive Charlottesville, VA 22904-4319 Andover, MA 01810 Melvin_Yu@eri.eisai.com Dr. Michael G. Thomas Department of Bacteriology Dr. Tin-Wein Yu. University of Wisconsin-Madison Department of Biological Sciences 777 Highland Avenue Louisiana State University Madison, WI 53705-2222 Baton Rouge, LA 70803-1715 yu@lsu.edu Copyright © 2005 CRC Press, LLC Contents Chapter 1 Introduction Gordon M. Cragg, David G. I. Kingston, and David J. Newman Chapter 2 Camptothecin and Its Analogs Nicolas J. Rahier, Craig J. Thomas, and Sidney M. Hecht Chapter 3 The Discovery and Development of the Combretastatins Kevin G. Pinney, Christopher Jelinek, Klaus Edvardsen, David J. Chaplin, and George R. Pettit Chapter 4 Homoharringtonine and Related Compounds Hideji Itokawa, Xihong Wang, and Kuo-Hsiung Lee Chapter 5 Podophyllotoxins and Analogs Kuo-Hsiung Lee and Zhiyan Xiao Chapter 6 Taxol and Its Analogs David G. I. Kingston Chapter 7 The Vinca Alkaloids Françoise Guéritte and Jacques Fahy Chapter 8 The Bryostatins David J. Newman Chapter 9 The Isolation, Characterization, and Development of a Novel Class of Potent Antimitotic Macrocyclic Depsipeptides: The Cryptophycins Rima S. Al-awar and Chuan Shih Chapter 10 Chemistry and Biology of the Discodermolides, Potent Mitotic Spindle Poisons Sarath P. Gunasekera and Amy E. Wright Chapter 11 The Dolastatins: Novel Antitumor Agents from Dolabella auricularia Erik Flahive and Jayaram Srirangam Copyright © 2005 CRC Press, LLC Chapter 12 Ecteinascidin 743 (ET-743; Yondelis™), Aplidin, and Kahalalide F Rubén Henríquez, Glynn Faircloth, and Carmen Cuevas Chapter 13 Discovery of E7389, a Fully Synthetic Macrocyclic Ketone Analog of Halichondrin B Melvin J. Yu, Yoshito Kishi, and Bruce A. Littlefield Chapter 14 HTI-286, A Synthetic Analog of the Antimitotic Natural Product Hemiasterlin Raymond J. Andersen and Michel Roberge Chapter 15 The Actinomycins Anthony B. Mauger and Helmut Lackner Chapter 16 Anthracyclines Federico Maria Arcamone Chapter 17 Ansamitocins (Maytansinoids) Tin-Wein Yu and Heinz G. Floss Chapter 18 Benzoquinone Ansamycins Kenneth M. Snader Chapter 19 Bleomycin Group Antitumor Agents Sidney M. Hecht Chapter 20 Biochemical and Biological Evaluation of (+)-CC-1065 Analogs and Conjugates with Polyamides Rohtash Kumar and J. William Lown Chapter 21 Epothilone, a Myxobacterial Metabolite with Promising Antitumor Activity Gerhard Höfle and Hans Reichenbach Chapter 22 Enediynes Philip R. Hamann, Janis Upeslacis, and Donald B. Borders Chapter 23 The Mitomycins William A. Remers Copyright © 2005 CRC Press, LLC Chapter 24 Staurosporines and Structurally Related Indolocarbazoles as Antitumor Agents Michelle Prudhomme Chapter 25 Combinatorial Biosynthesis of Anticancer Natural Products Michael G. Thomas, Kathryn A. Bixby, and Ben Shen Chapter 26 Developments and Future Trends in Anticancer Natural Products Drug Discovery David J. Newman and Gordon M. Cragg Copyright © 2005 CRC Press, LLC 1 Introduction Gordon M. Cragg, David G. I. Kingston, and David J. Newman The search for new lead compounds is a crucial element of modern pharmaceutical research. Natural products provided the only source of pharmaceuticals for thousands of years, and natural products have made enormous contributions to human health through compounds such as quinine, morphine, aspirin (a natural product analog), digitoxin, and many others. The potential of using natural products as anticancer agents was recognized in the 1950s by the U.S. National Cancer Institute (NCI) under the leadership of the late Dr. Jonathan Hartwell, and the NCI has since made major contributions to the discovery of new naturally occurring anticancer agents through its contract and grant support, including an important program of plant and marine collections. Many, although not all, of the compound classes described in this text owe their origin in whole or in part to NCI support. In spite of the success of the natural-products approach to anticancer drug discovery, as exemplified by the following chapters, in recent years their importance as a source of molecular diversity for drug discovery research and development has been overshadowed by various newer approaches currently in favor. These approaches include chemical ones which make heavy use of combinatorial chemistry, and biological ones such as manipulation of biosynthetic pathways of microbial metabolites through combinational biosynthetic techniques. It is thus worthwhile to review briefly the major reasons why the natural products are so important. First, there is a strong biological and ecological rationale for plants and marine invertebrates to produce novel bioactive secondary metabolites. The importance of plants and marine organisms as a source of novel compounds is probably related in large measure to the fact that they are not mobile and hence must defend themselves by deterring or killing predators, whether insects, fish, microorganisms, or animals. Plants and marine organisms have thus evolved a complex chemical defense system, which can involve the production of a large number of chemically diverse com- pounds. It has been proposed that all natural products have evolved to bind to specific receptors,1 and evidence for the advantage that natural products give an organism over predators has been found in ecological studies in the marine environment related to predator deterrence.2 As far as microbial species are concerned, secondary metabolites from the prokaryota and also from certain phyla in the eukaryota are actually synthesized in a combinatorial manner.3 This is the case because the genes that are ultimately responsible for the chemical structures obtained from these microbes, plants, and marine invertebrates can frequently be “shuffled” between taxa, and these shuffled genes produce diverse secondary metabolites, some of which are of use to man. Second, natural products have historically provided many major new drugs. Several recent reviews have provided data that document the importance of natural products as a source of bioactive compounds,3–6 and the conclusions can be summarized by a recent review that states, “In retrospect, the use of natural products has been the single most successful strategy in the discovery of modern medicines.”7 Copyright © 2005 CRC Press, LLC Third, natural products provide drugs that would be inaccessible by other routes. A large part of the reason for the importance of natural products in drug discovery is that natural products provide drugs that would be inaccessible by other routes. Thus, compounds such as paclitaxel (Taxol®) or halichondrin would never be prepared by standard “medicinal chemistry” approaches to drug discovery, even including the newer methods of combinatorial chemistry. Likewise, the new approach of combinatorial biosynthesis, although important, is unlikely in the near future to yield new compounds that have the complexity of bryostatin or camptothecin. The claim that synthetic compounds cannot match natural products for their structural diversity is borne out by a statistical study by Henkel and his collaborators, in which over 200,000 compounds from various databases were compared in various ways for their molecular diversity. The authors concluded “about 40% of the natural products are not represented by synthetic compounds” and that “the potential for new natural products is not exhausted and natural products still represent an important source for the lead-finding process.”8 Fourth, natural products can provide templates for future drug design. In many cases, the isolated natural product may not be an effective drug for any of several possible reasons, but it may nevertheless have a novel pharmacophore. Certainly, natural products, with their complex three-dimensional structures, are well suited as ligands for such targets as protein–protein interac- tions and for the selectivity needed to differentiate between the various protein kinases, to cite just two examples. Once a novel natural product chemotype has been discovered, it can be developed by medicinal chemistry or combinatorial chemistry into improved agents, as described in several chapters in this book. Examples of this are the drugs etoposide and teniposide, derived from the lead compound podophyllotoxin (Chapter 5); numerous analogs derived from Taxol (Chapter 6); topotecan, derived from camptothecin (Chapter 2); and the synthetic clinical candidates E7389 and HTI-286, developed from the marine leads halichondrin B and hemiasterlin, respectively (Chapters 14 and 15). In summary, the approach to drug discovery from natural sources has a historical justification (it has yielded many important new pharmaceuticals), a biochemical rationale (the position of marine organisms and plants in the ecosystem demands that they produce defense substances, many of which have a novel phenotype, and microbial species can produce new secondary metabolites by gene shuffling), and a chemical rationale (natural products provide templates for drug design). This book covers the major classes of clinically used anticancer natural products. The chapters are grouped by the source organism (plant, marine, or microbial), although it is recognized that the distinction is not always clear-cut, and some so-called marine natural products, for example, may in fact be produced by symbiotic microbial species (see, e.g., the discussion of bryostatin in Chapter 8). Within each chapter, the coverage normally includes the history of the drug and a discussion of its mechanism in action, its medicinal chemistry, its synthesis, and its clinical applications. The book concludes with a chapter on the increasingly important biosynthetic approaches to “unnatural natural products” and with a final chapter looking ahead to future devel- opments in anticancer natural products drug discovery. Because of the wealth of information available, the coverage in all of these chapters is of necessity selective rather than comprehensive, but the authors and editors have attempted to provide the most important recent results in each area. The editors hope that this volume will not only serve as a convenient summary of the current status of research and development of some of the most effective anticancer agents available today, but will also serve as an inspiration and a challenge to a new generation of scientists to engage in developing new and even better drugs from Nature’s bounty. REFERENCES 1. Williams, D.H. et al., Why are secondary metabolites (natural products) biosynthesized? J. Nat. Prod. 52, 1189, 1989. Copyright © 2005 CRC Press, LLC 2. Paul, V.J. Ecological Roles of Marine Natural Products. Cornell University Press, Ithaca, NY, 1992. 3. Kingston, D.G.I. and Newman, D.J. Mother Nature’s combinatorial libraries:

Their influence on the synthesis of drugs. Curr. Opin. Drug Disc. Dev. 5, 304, 2002. 4. Shu, Y.-Z. Recent natural products based drug development: A pharmaceutical industry perspective. J. Nat. Prod. 61, 1053, 1998. 5. Newman, D.J., Cragg, G.M., and Snader, K.M. Natural products as sources of new drugs over the period 1981–2002. J. Nat. Prod. 66, 1022, 2003. 6. Newman, D.J., Cragg, G.M., and Snader, K.M. The influence of natural products upon drug discovery. Nat. Prod. Rep. 17, 215, 2000. 7. Tulp, M. and Bohlin, L. (2002) Functional versus chemical diversity: Is biodiversity important for drug discovery? Trends Pharm. Sci. 23, 225, 2002. 8. Henkel, T., et al., Statistical investigation into the structural complementarity of natural products and synthetic compounds. Angew. Chem. Int. Ed., 38, 643, 1999. Copyright © 2005 CRC Press, LLC 2 Camptothecin and Its Analogs Nicolas J. Rahier, Craig J. Thomas, and Sidney M. Hecht CONTENTS I. Introduction II. Mechanism of Action A. Poisoning of Topoisomerase I B. Other Biochemical Effects of CPT III. Synthetic Studies A. Synthesis of Racemic CPT B. Asymmetric Synthesis C. Semisynthetic Methods IV. Medicinal Chemistry A. A/B/C/D Ring Analogs B. The E-Ring Lactone C. Water Soluble/Insoluble Analogs D. Conjugates V. Development of Clinically Useful Camptothecin Analogs A. Topotecan B. Irinotecan C. Analogs Currently under Evaluation VI. Conclusions VII. Acknowledgments References I. INTRODUCTION The antineoplastic agent camptothecin (CPT) (1) (Figure 2.1) was isolated from extracts of Camp- totheca acuminata by Wani and Wall.1 Preclinical studies revealed that CPT had remarkable activity against L1210 leukemia. The marginal water solubility of CPT encouraged researchers to initiate clinical investigation of the drug with the water-soluble sodium salt (2). Unfortunately, poor tumor suppression and numerous side effects were associated with treatment by 2; consequently, the trials were suspended.2 Interest in CPT was reestablished with the discovery that topoisomerase I (topo I) was the principal cellular target of the drug.3 Since this discovery, CPT has been the focus of numerous studies; to date, two analogs have been approved for clinical use — the semisynthetic, water-soluble analogs topotecan (Hycamtin) (3) and irinotecan (Camptosar) (4) (Figure 2.1). This chapter summarizes studies of the mechanism of action of CPT, as well as several of the synthetic studies, and provides an account of the current structure-activity studies. Also discussed briefly is the clinical development of topotecan, irinotecan, and several analogs that are currently in various stages of clinical trials. Copyright © 2005 CRC Press, LLC 9 7 10 5 A 4 O O B C N 16a N 13 N 1 3 D 6 17 N 14 15 E O OH 18 2 O- 1 Na+ 1 1 2 9 0 HO O 2 HO O N HO O N N N O N O O N O N HO O O 3 4 OH O FIGURE 2.1 Structures of camptothecin (1), the water-soluble sodium salt (2), topotecan (3), and irinotecan (4). II. MECHANISM OF ACTION A. POISONING OF TOPOISOMERASE I In 1985, it was reported that CPT stabilized the DNA–topoisomerase I covalent binary complex.3 It had previously been shown that CPT is capable of inhibiting DNA synthesis, thereby causing cell death during the S-phase of the cell cycle.4 The S-phase specific cytotoxicity is directly correlated to the occurrence of irreversible DNA cleavage when the replication fork encounters the covalent DNA–enzyme binary complex (Figure 2.2).5 It has been demonstrated that deletion of the gene for topo I from Saccharomyces cerevisiae results in viable cells that are fully resistant to CPT.6 Further, a number of CPT-resistant cell lines have been identified, each containing mutations within topo I.7 These studies clearly support a mechanism of action for CPT involving topo I–mediated DNA cleavage. The topoisomerases (type I and type II) are a class of enzymes that mediate the relaxation of chromosomal DNA prior to DNA replication and transcription.8 Mechanistically, topo II effects this relaxation via transient double-strand cleavage, DNA strand passage, and religation of the phosphodiester backbone. This process requires ATP and alters the DNA linking number by multiples of two. The topo I mechanism involves energy-independent single-strand DNA cleavage, followed by strand passage and religation.9 The mechanism of topo I–mediated DNA relaxation is known to involve an active site tyrosine that cleaves DNA by nucleophilic attack of the active site tyrosine phenolic OH group on the phosphodiester backbone (Figure 2.3). The resulting DNA–topo I intermediate is a covalent binary complex. The cleaved DNA strand can be passaged around the unbroken DNA strand; the intact duplex is reformed on religation of the phosphodiester bond, with the concomitant release of topo I. Although topo I may be capable of DNA cleavage at a number of sites, it exhibits a strong preference for the nucleoside thymidine as the nucleobase directly upstream (the -I position) (cf. Figure 2.3). Stabilization of the covalent binary complex by CPT has been noted to involve an additional preference for guanosine at the +1 position. Most of the agents that poison type I and type II topoisomerases are characterized by their ability to inhibit the religation step during DNA relaxation (Figure 2.3). Topo II poisons include a Copyright © 2005 CRC Press, LLC topo I CPT DNA CPT stabilization of the covalent binary complex DNA replication irreversible DNA cleavage cell death FIGURE 2.2 S-phase specific cytotoxic mechanism of camptothecin. FIGURE 2.3 Mechanism of DNA relaxation by human topoisomerase I. Copyright © 2005 CRC Press, LLC Major Groove H N O N H O C HO O G+1 H O N N Non-cleaved N O N Scissile A O T-1 O O H OH O Minor Groove Asp 533 FIGURE 2.4 Two-dimensional representation of the x-ray crystal structure of topotecan within the covalent binary complex. number of well-characterized clinical agents such as amsacrine and etopside.10 Several inhibitors of topo I have also been identified; however, CPT remains the most widely studied of this class of medicinal agents. The development of a precise understanding of the way in which CPT stabilizes the DNA–topo I covalent binary complex, and thereby inhibits the religation of duplex DNA, is an important current goal. CPT has no binding affinity for topo I, and only the positively charged CPT analog topotecan (3) has shown any DNA binding.11,12 However, the ability of CPT to bind to the covalent binary complex formed between topo I and DNA is sufficient to inhibit religation. There is compelling evidence that CPT is capable of interacting with the covalent binary complex at or near the interface between DNA and topo I. Hertzberg and coworkers have established that a CPT analog containing a bromoacetamide at carbon 10 was, on prolonged exposure, capable of forming a drug–enzyme crosslink.13 Pommier and coworkers later showed that a 7-chloromethylated CPT analog alkylated N3 of the guanine at the +1 cleavage site of DNA.14 A number of computational models have been formulated depicting the interaction between CPT and the covalent binary complex.15–17 These models posit different energy-minimized inter- actions between CPT, the DNA 5-TpG-3 base pair, and selected topo I amino acid residues known to be important based on biochemical studies or their proximity to the putative CPT binding site.18–21 The analysis of several CPT-resistant cell lines has provided important information regarding the amino acid residues that play a role in CPT binding.7 For example, mutational analysis of Asp 533, Arg364, Asn722, and Lys532 has revealed that each of these amino acid residues likely play a role in CPT binding.19–21 Staker et al. have recently reported the X-ray crystal structure of a ternary complex formed between DNA, topo I, and topotecan (3).22 The reported complex used a DNA oligonucleotide containing a 5-bridging phosphothioate to facilitate crystal formation and indicated that topotecan bound the covalent binary complex in an intercalative fashion (Figure 2.4). The only direct drug–enzyme hydrogen bond interaction was between Asp533 and the 20-hydroxyl group of topotecan. In addition, one hydrogen bond was observed with a water molecule. Carbons 7, 9, and 10 of CPT were positioned in a manner that situated them in the vicinity of the major groove. B. OTHER BIOCHEMICAL EFFECTS OF CPT It is generally accepted that the basis for CPT-induced cytotoxicity is contingent on CPT acting as a topo I poison (as opposed to an inhibitor of enzyme activity, per se).3–7 However, the antitumor selectivity of CPT is somewhat surprising given that topo I is an enzyme found in all cell types. Elevated levels of topo I are present in tumors of the colon, ovary, and prostate, which may explain the therapeutic index of CPT.23 Deficiencies in DNA repair capabilities in some cancer cells may provide another possible basis for cancer cell selectivity. The selective inhibition of all dividing Copyright © 2005 CRC Press, LLC cell populations represents another possible source of antitumor selectivity. Further, other effects of CPT exposure have been noted and merit discussion. Kauh and Bjornsti, using a genetic screen, have identified six dominant suppressors of camp- tothecin toxicity at a single genetic locus (SCT1).24 Mutant SCT1 cells were shown to express wild- type topo I, indicating additional factors in the overall cellular response to CPT. One report indicated that irinotecan, but not topotecan, inhibits acetylcholinesterase activity.25 Additional reports indicate that CPT activates the transcription factor NFκB, which has been implicated in numerous activities in vivo.26 Importantly, activation of NFκB has been implicated in the overproduction of interferons, triggering several cellular responses.26–29 In addition to its role in relaxation of supercoiled DNA, topo I is able to regulate transcription,30 recognize and cleave mismatched nucleotides at intrinsic cleavage sites,31 and associate with numerous proteins in vivo. Tazi and coworkers have reported that topo I is capable of influencing gene splicing by acting as a phosphorylating enzyme for SR proteins.32 This kinase activity is inhibited by CPT despite the fact that it has been shown to be unconnected to its DNA relaxation activity through mutational studies.33 Analysis of a family of topoisomerase I related function proteins (TRFp) demonstrate that one member of this family, TRF4p, plays a critical role in mitotic chromosome condensation during the S phase of the cell cycle.34 TRF4p is associated with the DNA binding protein Smc1p during chromosomal condensation, and reports detailing mutations to TRF4p have produced cell lines with unexpected hypersensitivity to CPT.35 III. SYNTHETIC STUDIES Given the finite supplies of natural CPT and the need to develop additional analogs, much effort has focused on practical synthetic routes to CPT and its analogs. The following are descriptions of several of the synthetic approaches to CPT and of the semisynthesis of topotecan (3) and irinotecan (4). A. SYNTHESIS OF RACEMIC CPT Following the initial publication of the structure of CPT, a number of synthetic strategies for the preparation of CPT were reported. The first total synthesis of (R,S)-CPT was described by Stork and Schultz in 1971.36 Numerous successful synthetic approaches to 20(R,S)-CPT have since been published and reviewed.37,38 Given the early availability of methods for racemic synthesis, relative to asymmetric approaches, research also focused on the chiral resolution of the racemates or key synthetic intermediates. In 1975, Corey and coworkers were the first to report the successful resolution of a chiral intermediate, leading to the preparation of 20(S)-CPT.39 Wani et al. and Teresawa et al. have also reported the successful resolution of the intermediates in the synthesis of CPT, using (R)-(+)-α-methyl-benzylamine.40,41 B. ASYMMETRIC SYNTHESIS Ejima et al. reported the first stereocontrolled synthesis of CPT via a diastereoselective ethylation process using N-tosyl-(R)-proline (6) (Scheme 2.1).42 Indolizine 5, the CD-ring precursor of the parent alkaloid, was employed in an approach to construction of ring E. Bromination of 5 followed by treatment with N-tosyl-(R)-proline (6) in the presence of base afforded compound 7. A diaste- reomeric mixture of (S,R)-8 and (R,R)-9 was quantitatively prepared by facial differentiated ethy- lation of 7 in 82% de; selective recrystallization provided pure 8 in 56% yield. Following Raney Ni catalyzed reduction of 8 and subsequent treatment with NaNO2, the optically pure ester 10 was obtained in 74% yield. The triester 10 was hydrolyzed using LiOH and lactonized to provide hydroxy lactone 11 having the proper 20(S)-configuration in 90% yield. Hydrolysis of the ketal Copyright © 2005 CRC Press, LLC SCHEME 2.1 functionality gave optically pure key intermediate 12, which was converted to 20(S)-CPT by Friedländer reaction with 2-aminobenzaldehyde derivative 13 in 84% yield. Comins and coworkers have successfully employed chiral auxiliaries to establish the

correct stereochemistry for the 20(S)-hydroxyl group (Scheme 2.2).43 Refinement of their method has culminated in the asymmetric synthesis of 20(S)-CPT in only six steps, using the α-ketobutyric ester derived from (-)-trans-2-(α-cumyl)cyclohexanol [(-)-TCC] (17) as chiral auxiliary (Scheme 2.2).44 Treatment of commercially available 2-methoxypyridine (14) with mesityllithium, followed by addition of N-formyl-N,N′,N′-trimethylethylenediamine, effected the alkylation of the aromatic ring at C3. Addition of n-BuLi followed by iodine and workup with aqueous NaBH4/CeCl3 provided alcohol 15 in 46% yield via a one-pot process. Conversion of 15 directly to 1,3-dioxane 16 using NaI/TMSCl/paraformaldehyde was accomplished in 87% yield. The DE ring precursor 18 was fashioned via another one-pot process involving lithium–halogen exchange effected with n-BuLi, followed by addition of chiral auxiliary 17. Addition of HCl effected protonation, acetal hydrolysis, and lactonization to give intermediate 18 in 60% yield (93% ee). Coupling of 18 with the quinoline intermediate 19 was accomplished via displacement of the primary iodide to provide enantiopure 20 in 81% yield. The C-ring was closed using a Heck reaction through treatment of 20 with Pd(II) and potassium acetate to provide 20(S)-CPT in 64% yield. 1) MesLi 1) n-BuLi O O 2) 2) N N CHO O O O CO2R* 17 N N OH TMSCl/NaI N 3) n-BuLi (CH20)n 3) H+ 4) I2 I I 14 15 16 5) NaBH4 R*=(-)-TCC O O H N O t-BuOK N (Ph3P)2Pd(OAc)2 N Cl 1 O I O KOAc HO N Cl 18 HO O 19 20 SCHEME 2.2 Copyright © 2005 CRC Press, LLC OMe 1) etherification OMe (DHQD)2-Pyr O I OMe N CHO N O K3Fe(CN)6 2 N O HCl HN O 1 I 2) K Heck 2OsO2(OH)4 CaCO3 O O K 2 steps cyclization 2CO3 OH OH MeSO 21 2NH 22 2 23 18 SCHEME 2.3 Two other research groups have employed chiral auxiliaries to establish the S-configuration at the C20 position of CPT. Tagami et al. used a Davis reagent, (2R, 8aS)-(+)-(camphorylsulfonyl)oxaziridine), to asymmetrically hydroxylate 20-deoxycamptothecin.45 In 2002, Bennasar et al. made use of (2R,5R)- 2-tert-butyl-5-ethyl-1,3-dioxolan-4-one to establish C20 asymmetry and synthesize 20(S)-CPT.46 To control absolute stereochemistry at C20, Ciufolini and Roschangar reported a synthesis of 20(S)-CPT that made use of an aldehyde intermediate obtained by an enzymatic desymmetrization of a corresponding malonate.47 In 1998, Imura and coworkers described the first asymmetric synthesis of a key chiral intermediate, using enzyme-catalyzed resolution.48 Fang et al. applied the first chiral catalytic method to prepare 20(S)-CPT (Scheme 2.3).49 Reductive etherification of aldehyde 21 followed by intramolecular Heck reaction gave the cyclic enol ether 22 in 45% yield (Scheme 2.3). Sharpless asymmetric dihydroxylation of 22 using (DHQD)2-Pyr as the chiral catalyst followed by oxidation with iodine and CaCO3 was performed to synthesize the DE ring precursor 23 with 94% ee (90% yield). Treatment of 23 with HCl provided the enatiomerically pure pyridone 18 in 74% yield. The authors completed the synthesis of 20(S)- CPT using the Comins route (cf Scheme 2.2).43,44 Jew et al. made further use of the catalytic asymmetric Sharpless dihydroxylation using (DHQD)2-Pyr as chiral catalyst, resulting in stereocontrolled oxidation of carbon 20 in greater than 90% ee in the total synthesis of 20(S)-CPT.50 In 2002, Blagg and Boger established the configuration of the C20 (S) tertiary alcohol through a Sharpless asymmetric dihydroxylation reaction, using a 3,4,5-trimethoxyphenyl-derived DHQ dimer ligand in 86% ee.51 Curran and coworkers have reported a synthesis of 20(S)-CPT based on a 4+1 radical cascade annulation (Scheme 2.4).52 Lactone 24 was obtained in a fashion similar to the synthesis developed by Fang et al.49 Exchange of the TMS group in 24 for iodine, followed by demethylation, provided 25 in 33% yield (Scheme 2.4). N-Propargylation of lactone 25 provided alkyne 26 in 88% yield followed by isonitrile treatment under irradiation to provide 20(S)-CPT in 63% yield. Curran and coworkers have subsequently reported improvements in the synthesis of the enantiopure DE ring precursor 25, using a samarium catalyst.53 Radical methods of this type have been shown to tolerate A- and B-ring substituents and have, therefore, been used for the synthesis of numerous CPT analogs.52 C. SEMISYNTHETIC METHODS In addition to these total syntheses, many synthetic efforts have been focused on producing analogs of CPT. The majority of these efforts have involved the semisynthetic manipulation of CPT. The approaches to irinotecan and topotecan are described. Sawada et al. first reported the synthesis of irinotecan (4) in 1991 (Scheme 2.5).54 Hydrogen peroxide was added to a solution of 20(S)-CPT in aqueous sulfuric acid in the presence of ferrous OMe O O 1) ICl H N O N O Br PhNC N O 1 Me3Si O 2) TMSI I O NaH, LiBr I O Me6Sn2 OH H2O cat. OH OH 24 25 26 SCHEME 2.4 Copyright © 2005 CRC Press, LLC Cl CH N N 3CH2CHO FeSO4, H2O2 O HO H irradiation O O N 2O2 O 1 N N 4 aq.H2SO4 N N+ H2SO4 N O O- O O 27 HO O 28 HO O 29 HO O SCHEME 2.5 sulfate and propionaldehyde to afford 7-ethyl CPT (27) in 77% yield (Scheme 2.5). 7-Ethyl CPT (27) was converted to the corresponding N-oxide 28 using hydrogen peroxide in acetic acid. Irradiation of 28 in acidic media furnished the active metabolite of irinotecan (SN-38) (29) in 49% yield. Treatment of SN-38 (29) with 4-(1-piperidino)-1-(piperidino)-chloroformate then provided irinotecan (4) in 80% yield. The second currently marketed CPT derivative, topotecan, was synthesized in 1991 by Kings- bury et al. in two steps starting from 20(S)-CPT (Scheme 2.6).55 Conversion of 20(S)-CPT to 10- hydroxy CPT (30) was accomplished through a reduction-oxidation sequence in 71% yield (Scheme 2.6). Treatment of 30 with dimethylamine in aqueous formaldehyde and acetic acid provided topotecan (3) in 62% yield. IV. MEDICINAL CHEMISTRY Synthetic and semisynthetic analogs of CPT have provided the means to study the relationship between CPT structure and function. A clear understanding of the mechanism by which CPT inhibits DNA religation and insight into the exact roles of the various structural elements of CPT are essential to preparing novel, therapeutically important derivatives. At present, much is known regarding the SAR profile of CPT, and the following text is a summary description of innovative and important derivatives.38 This discussion focuses on the significance of the individual rings within the pentacyclic ring system, as well as the lone stereocenter. Further, we will consider analogs with substitutions, additions, and deletions within each of the five rings of CPT and specific derivatives that increase water solubility and, conversely, lipophilicity; and, finally, we will present an account of several CPT conjugates. A. A/B/C/D RING ANALOGS Alterations to the aromatic core of CPT are normally confined to substitutions on the A/B quinoline ring system. There is general agreement that the planarity of the four ring aromatic core is a requirement for topo I inhibition, as illustrated by studies that alter both the quinoline ring and the C/D ring systems.56-58 Additions to the C/D ring system are restricted by the lack of accessible carbons (limited to C5 and C14), and analogs with additions at those sites have generally displayed diminished activity. Exceptions include the C5 substituted 2-fluoroethyl ester analog and 5-eth- ylidene CPT, which displayed various degrees of in vitro and in vivo activity.59,60 N HO O 1) AcOH, H , HO 2/PtO2 1 atm N CH2O, Me2NH O 1 N N 2) Pb(OAc)4, AcOH N O O 30 OH O 3 HO O SCHEME 2.6 Copyright © 2005 CRC Press, LLC F O N N A B C N HO F N D N O E O B A B O A O HO irinotecan (4) topotecan (3) diflomotecan (38) O NH A B C N 2 HO N D A B E O A B 1 F N SN-38 (29) HO O exatecan (34) N t-Bu N SiMe3 O N NH N 2 NO2 O B A B A A A B B N O N N N CKD-602 lurtotecan 9-AC 9-NC karenitecin gimatecan (36) (33) (31) (32) (37) (35) FIGURE 2.5 Structure of selected camptothecin analogues. The vast majority of studies involving CPT modification have focused on additions to the quinoline ring system.38,61–63 Studies by Wani, Wall, and coworkers described a number of CPT analogs that established several broadly applicable principles regarding CPT structure and func- tion.64–67 Among these principles were that substitutions at carbons 11 and 12 were generally unfavorable, albeit with some exceptions, whereas substitutions at positions 7, 9, and 10 were generally accepted without significant loss of function. In addition, a study by Kingsbury and coworkers illustrated the accuracy of these observations.55 Included within these studies were the first descriptions of the clinically significant derivatives 9-aminoCPT (31), 9-nitroCPT (32) and topotecan (3) (Figure 2.5). B. THE E-RING LACTONE Although additions to the quinoline ring have been useful for preparing functional CPT analogs, alterations of the E-ring lactone have proven to be less successful. The two functional groups of the E-ring, that is, the 20(S)-hydroxyl group and the lactone, are both highly sensitive to alterations. The 20(R) enantiomer, which is accessible through synthetic methods, is inactive as both a topo I poison and an antitumor agent.68,69 The inactivity of the 20(R)-CPT argues for a remarkably specific interaction between the 20-hydroxyl group and the topo I–DNA covalent binary complex. Replace- ment of the 20(R) hydroxyl group with an amine results in a CPT analog with largely diminished activity, whereas 20-halogeno derivatives maintained substantial potency.69 The deoxy species is capable of inhibiting topo I–induced rearrangement of DNA but is inactive as a topo I poison and produces no cytotoxicity.69 A notable difference between these 20-substituents involves their ability to participate in hydrogen-bonding interactions. In the aggregate, these studies suggest that the role of the 20-hydroxyl group may be that of a specific hydrogen bond acceptor, rather than a donor. Alternatively, a 20-substituent may be required to maintain a conformation of ring E conducive to ternary complex stabilization. Copyright © 2005 CRC Press, LLC The E-ring lactone is also important for maintaining the potency of CPT as a topo I poison and cytotoxic agent. The apparent inactivity of the carboxylate form of CPT (2) led researchers to consider the more hydrolytically stable lactam derivative.70 Surprisingly, although the CPT- lactam did, in fact, enhance the stability of the E-ring in water-based assays, it was ineffectual as a topo I poison. Likewise, the carbinol lactam, thiolactone, and the imide derivatives were also essentially inactive.70 Unsurprisingly, the α-hydroxylactone ring exists in an equilibrium that favors the inactive carboxylate form at physiological pH.70 Further, there is a strong preference of human serum albumin (HSA) to bind the carboxylate form of CPT, which could plausibly constitute an obstacle in delivering the active form of the drug.71 Although the lactam derivative was unsuccessful at overcoming this difficulty, the replacement of the α-hydroxylactone ring with a β-hydroxylactone ring resulted in a class of CPT analogs, designated homo-CPT, with increased plasma stability and good inhibitory activity.72 In spite of the obvious importance of the E-ring lactone to the topoisomerase I inhibitory activity, Cagir et al. have recently reported that the naturally occurring pyrroloquinazolinoquinoline alkaloid luotonin A functions as a topoisomerase I poison in much the same fashion as CPT.73 Luotonin A is identical with CPT in rings A–C and differs mainly in the E-ring which is an unsubstituted benzene ring in luotonin A.73 C. WATER SOLUBLE/INSOLUBLE ANALOGS The success of topotecan and irinotecan in the clinic has prompted the evaluation of other water- soluble analogs of CPT. Numerous examples exist, and studies highlight both the biochemical activity of these derivatives and the increased stability of the E-ring lactone.38 Among the most potent of these analogs is 10,11-(methylenedioxy)-7-((N-methylpiperazino)methyl)CPT (lurtote- can) (33) (Figure 2.5), which is reported to be a more effective cytotoxic agent than topotecan.74 Exactecan (34) (Figure 2.5) is another highly water soluble analog that has received significant attention. The potency of exatecan has been reported to be up to 28-fold greater than that of topotecan toward various human malignant cells.75 The required integrity of the E-ring lactone in comparison with the preference of HSA to bind the carboxylate form of CPT has also prompted researchers to examine analogs with increased lipophilicity. Burke and coworkers, using silicon-containing derivatives of CPT, have prepared a number of highly active analogs, designated silatecans.76 Similarly, Zunino and coworkers have used aminomethyl, iminomethyl, and oxyiminomethyl substitutions as an

alter- native method to increase the lipophililic nature of CPT.77 Two of the most successful of these analogs are 7-(tert-butyldimethylsilyl)-10-hydroxyCPT (DB-67) and 7-(tert-butoxyiminome- thyl)CPT (Gimatecan)(35) (Figure 2.5), which have both shown high levels of cytotoxicity.77,78 Curran, Burke, and coworkers have also demonstrated the success of a silatecan/homoCPT derivative (Du1441) that has demonstrated the highest level of lactone stability yet reported.79 The success of these water-insoluble derivatives, in conjunction with liposomal core loading of the drug, constitutes a powerful tool for the delivery of CPT in the E-ring lactone form. D. CONJUGATES The development of CPT conjugates has been successful in affording CPT analogs having novel properties.38 Two strategies have been employed for the preparation of these conjugates. The first uses the 20(S)-hydroxyl group as a conjugation point, and the second relies on semisynthetic functional groups appended to the quinoline ring (e.g. amino, hydroxyl, and carboxylic acid groups). Several studies have produced derivatives of potential therapeutic importance. Firestone and coworkers have reported the elaboration of an immunoconjugate derived from CPT and the tumor-specific antibody BR96.80 The conjugation was achieved using a carbamate Copyright © 2005 CRC Press, LLC linkage that can be cleaved by cathespin B. The conjugate was designed to eliminate dose-limiting side effects, and preliminary in vitro activity was reported to be superior to that of CPT alone. The CPT-induced sequence-specific cleavage of DNA is limited to TpG sequences and, accord- ingly, lacks the wherewithal to be used in an antisense-type approach of targeting specific genetic sequences. Conjugates involving triplex-forming oligonucleotides (TFOs) covalently attached to campothecin through amide linkages with various aliphatic spacers have extended the sequence specificity of CPT while maintaining the potency of the drug.81 Wang and Dervan have extended the generality of this strategy by using the minor groove binding hairpin polyamide within CPT conjugates.82 Greenwald and coworkers have studied the use of CPT-PEG prodrugs as a mechanism for delivery of CPT solid tumors.83 Specifically, the preparation of glycine- and alanine-based linkers in PEG-β-CPT was demonstrated as a successful strategy at the levels of biodistribution and in vivo potency.83 Recently, Greenwald et al. have reported the preparation and high in vivo activity of two PEG-CPT derivatives conjugated through the open carboxylate form of CPT.84 V. DEVELOPMENT OF CLINICALLY USEFUL CAMPTOTHECIN ANALOGS CPT is not used as an anticancer agent because of its poor water solubility and numerous toxicities. Two CPT derivatives, topotecan (3) and irinotecan (4), are used in several countries for the treatment of solid tumors. Further, there are a number of new CPT analogs in various stages of clinical trials as well as expanding clinical development of CPT analogs involving combination regimens. The clinical applications of the CPT analogs have been discussed in several excellent reviews, and a brief summary is detailed here.85,86 A. TOPOTECAN Topotecan (3) (Hycamtin, GlaxoSmithKline) is a semisynthetic derivative of CPT with a basic N,N-dimethylaminomethyl functional group at C9 that confers improved water solubility to the molecule. Topotecan was approved by the U.S. Food and Drug Administration in 1996 and is presently used as second-line therapy for advanced ovarian cancer in patients, following unsuc- cessful treatment with platinium-based therapeutics or paclitaxel-containing chemotherapy regi- mens. It is also approved as a therapeutic option for recurrent small-cell lung cancer.85 Topotecan is most commonly administered as an intravenous infusion. The biological half-life of topotecan in humans is much shorter than that of other CPT derivatives, and as a consequence, drug accumulation does not occur. The fraction of topotecan bound to plasma proteins is also much lower than that of other CPT derivatives. Neutropenia has proven to be the most encountered dose-limiting toxicity; however, a degree of thrombocytopenia is often associated with topotecan treatment as well. In addition to its action on ovarian and small-cell lung cancers, topotecan has shown activity against hematological malig- nancies. Topotecan combination regimens with paclitaxel, etoposide, cisplatin, cytarabine, and cyclophosphamide, as well as with other treatment modalities such as radiation therapy, are in development.86 B. IRINOTECAN Irinotecan (4) (Camptosar, Pfizer) is a water-soluble prodrug designed to facilitate parenteral administration of the potent 7-ethyl-10-hydroxycamptothecin analog SN 38 (29). It contains a dibasic bispiperidine moiety, linked through a carbonyl group to a 10-hydroxy functionality. Enzy- matic cleavage of the bispiperidine functionality by carboxylesterases predominantly located in the liver affords SN-38, the biologically active compound, which is 1000-fold more potent as an Copyright © 2005 CRC Press, LLC inhibitor of purified topoisomerase I in vitro than irinotecan. Irinotecan was approved in 2000 by the U.S. Food and Drug Administration for use in the treatment of advanced colorectal cancer, both as first-line therapy in combination with 5-fluorouracil and as salvage treatment in 5-fluorouracil refractory disease. Irinotecan is most commonly administered as an intravenous infusion.85 The biological half-life of the lactone form of SN-38 exceeds that of topotecan, which represents a potential pharmacological advantage. Further, in comparison with other CPT analogs, a relatively large percentage of the intact lactone form of both irinotecan and SN-38 persists in the plasma of patients after drug administration, attributable to the preferential binding of irinotecan to serum albumin in its lactone form. The principal dose-limiting toxicity of irinotecan is delayed diarrhea, with or without neutro- penia. Promising antitumor activity has also been observed against small-cell and non-small-cell lung cancer, ovarian cancer, and cervical cancer and in recent clinical trials involving patients with malignant gliomas. Studies to evaluate additional irinotecan drug combinations with taxanes, anthracyclines, vinca alkaloids, or alkylating agents are in progress. C. ANALOGS CURRENTLY UNDER EVALUATION Exatecan mesylate dihydrate (DX-8951f) (34) (Daiichi Pharmaceuticals) is a synthetic hexacyclic water-soluble analog of CPT that inhibits the growth of human tumor cell lines in vitro and that of tumor xenografts in vivo, including tumors resistant to topotecan and irinotecan.85 Reports of phase II clinical trials indicate that exatecan may have significant activity against small-cell and non-small-cell lung cancer, hepatocellular cancer, colorectal cancer, and pancreatic carcinoma.85-88 CKD-602 (36) (Chong Kun Dang Pharmaceuticals) is a potent topo I inhibitor that overcomes the poor aqueous solubility and toxicity profile of CPT.87 This compound shows enhanced activity against a series of cell lines in vitro and against L1210 in vivo.88 Furthermore, partial responses were observed in patients with stomach and ovarian cancer. CKD-602 is presently in phase II clinical trials.87 NX211 (NeXstar Pharmaceuticals) is liposomal formulation of lurtotecan (33) that prolonged the systemic duration of lurtotecan and increased its therapeutic index. In phase I clinical trials, tumor responses were observed in patients with ovarian cancer.85-88 9-AC (IDEC-132) (31) (National Cancer Institute) is a semisynthetic CPT analog with potent antitumor activity against a wide spectrum of human tumor xenograft models.85 Phase II studies using intravenous infusion have been conducted in patients with various type of malignancies with disappointing results. A solid oral dosage form was developed by incorporating 9-AC into poly(ethylene)glycol-1000, which yielded bioavailabilities of 49% in cancer patients. Thus, the oral bioavailability of 9-AC in cancer patients is far superior to that observed with any other CPT derivative to date. Phase II studies using this formulation have been initiated in Europe.85 9-NC (Rubitecan) (32) (SuperGen) is a potent but poorly soluble CPT analog developed exclu- sively for oral administration.85 This compound is metabolically converted in vivo into 9-AC. Rubite- can is more robust and relatively inexpensive to prepare compared to 9-AC.86 It is in phase III clinical trials in pancreatic cancer as well as phase II clinical testing for the treatment of 11 additional tumor types.87 An aerosol delivery of liposomal 9-NC has been employed in phase I clinical studies.86 Karenitecin (BNP-1350) (37) (Bionumerik Pharmaceuticals) is a semisynthetic, lipophilic compound that exhibits more potent cytotoxic activity than CPT both in vitro and in vivo, with enhanced oral bioavailability. The in vitro assays for antiproliferative capacity in colon cancer cell lines indicated that Karenitecin was similar in effectiveness to SN-38. Karenitecin has com- pleted phase I clinical trials for pancreatic and colorectal cancer, and it is undergoing phase II clinical trials.87 Gimatecan (35) (ST1481) (Sigma-Tau and Italian National Cancer Institute) is a semisynthetic, lipophilic 7-oxyiminomethylCPT. Gimatecan showed cytotoxicity against a panel of human tumor xenografts and is undergoing phase I clinical trials.88 Copyright © 2005 CRC Press, LLC Diflomotecan (BN-80915) (38) (Beaufour Ipsen) is a homoCPT analog that demonstrates enhanced E-ring stability. Diflomotecan exhibited high antiproliferative activity in a panel of tumor cell lines, including those exhibiting multidrug resistance, and was found to be active at very low doses in a variety of human tumor xenografts. BN-80915 is currently undergoing phase I clinical evaluation in Europe.86–89 In addition, efforts to optimize delivery strategies for CPT derivatives such as polymer conju- gation, nanoparticule encapsulation, and liposomal core loading are under way.90 VI. CONCLUSIONS Camptothecin and its analogs represent an exciting class of antineoplastic agents targeted at numerous types of cancers. At present there exists considerable interest in CPT, and studies continue to offer meaningful insights into the drug nearly 40 years after Wani and Wall first reported its pentacyclic structure. Today the biochemical basis of CPT activity is better defined, allowing scientists to focus their investigations more effectively. Recent advances in the synthesis of CPT and its analogs are such that the construction of new analogs is achievable on a scale not possible even a decade ago. Development of novel analogs that optimize and exploit important structural features and further expand the therapeutic potential of CPT is being reported. VII. ACKNOWLEDGMENTS We thank Dr. Cyrille Gineste, University of Virginia, for helpful discussions during the preparation of this review. Work in our laboratory has been supported by National Institutes of Health research grant CA78415, awarded by the National Cancer Institute, and by American Cancer Society fellowship PF-02-090-01-CDD (to C.J.T.). REFERENCES 1. Wall, M.E. et al., Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata, J. Am. Chem. Soc., 88, 3888, 1966. 2. Moertel, C.G. et al., Phase II study of camptothecin (NSC-100880) in the treatment of advanced gastrointestinal cancer, Cancer Chemother. Rep., 56, 95, 1972. 3. Hsiang, Y.-H. et al., Camptothecin induces protein-linked DNA breaks via mammalian DNA topoi- somerase I, J. Biol. Chem., 260, 14873, 1985. 4. Gallo, R.C., Whang-Peng, J., and Adamson, R.H., Studies on the antitumor activity, mechanism of action, and cell cycle effects of camptothecin, J. Natl. Cancer Inst., 46, 789, 1971. 5. Hsiang, Y.-H., Lihou, M.G., and Liu, L.F., Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complex as a mechanism of cell killing by camptothecin, Cancer Res., 49, 5077, 1989. 6. Nitiss, J. and Wang, J.C., DNA topoisomerase-targeting antitumor drugs can be studied in yeast, Proc. Natl. Acad. Sci. USA, 85, 7501, 1988. 7. Pommier, Y. et al., Topoisomerase I inhibitors: selectivity and cellular resistance, Drug Resistance Updates, 2, 307, 1999. 8. Champoux, J.J., DNA topoisomerases: structure, function, and mechanism, Annu. Rev. Biochem., 70, 369, 2002. 9. Pommier, Y. et al., Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme, Biochim. Biophys. Acta, 1400, 83, 1998. 10. Sengupta, S.K., Topoisomerase II inhibitors, in Cancer Chemotherapeutic Agents, Foye, W.O., Ed., American Chemical Society, Washington D.C., 1995, pp. 205–292. 11. Hertzberg, R.P., Caranfa, M.J., and Hecht, S.M., On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex, Biochemistry, 28, 4629, 1989. Copyright © 2005 CRC Press, LLC 12. Yang, D. et al., DNA interactions of two clinical camptothecin drugs stabilize their active lactone forms, J. Am. Chem. Soc., 120, 2979, 1998. 13. Hertzberg, R.P. et al., Irreversible trapping of the DNA-topoisomerase I covalent complex, J. Biol. Chem., 265, 19287, 1990. 14. Pommier, Y. et al., Interaction of an alkylating camptothecin derivative with a DNA base at topoi- somerase I-DNA cleavage sites, Proc. Natl. Acad. Sci. USA, 92, 8861, 1995. 15. Fan, Y. et al., Molecular modeling studies of the DNA-topoisomerase I ternary cleavable complex with camptothecin, J. Med. Chem., 41, 2216, 1998. 16. Kerrigan, J.E. and Pilch, D.S., A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin, Biochemistry, 40, 9792, 2001. 17. Laco, G.S. et al., Human topoisomerase I inhibition: docking camptothecin and derivatives into a structure-based active site model, Biochemistry, 41, 1428, 2002. 18. Redinbo, M.R. et al., Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA, Science, 279, 1504, 1998. 19. Yoshimasa, U. et al.,

Characterization of a novel topoisomerase I mutation from a camptothecin- resistant human prostate cancer cell line, Cancer Res., 61, 1964, 2001. 20. Fujimori, A. et al., Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin, Cancer Res., 55, 1339, 1995. 21. Jensen, A.D., and Svejstrup, J.Q., Purification and characterization of human topoisomerase I mutants, Eur. J. Biochem., 236, 389, 1996. 22. Staker, B.L. et al., The mechanism of topoisomerase I poisoning by a camptothecin analog, Proc. Natl. Acad. Sci. USA, 99, 15387, 2002. 23. Husain, I. et al., Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity and implications for cancer chemotherapy, Cancer Res., 54, 539, 1994. 24. Kauh, E.A. and Bjornsti, M.-A., SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I, Proc. Natl. Acad. Sci. USA, 92, 6299, 1995. 25. Kawato, Y. et al., Inhibitory activity of camptothecin derivatives against acetylcholinesterase in dogs and their binding activity to acetylcholine receptors in rats, J. Pharm. Pharmacol., 45, 444, 1993. 26. Siddoo-Atwal, C., Haas, A.L., and Rosin, M.P., Elevation of interferon beta-inducible proteins in ataxia telangiectasia cells, Cancer Res., 56, 443, 1996. 27. DeSai, S.D. et al., Ubiquitin, SUMO-1, and UCRP in camptothecin sensitivity and resistance, Ann. N. Y. Acad. Sci., 922, 306, 2000. 28. Ohwada, S. et al., Interferon potentiates antiproliferative activity of CPT-11 against human colon cancer xenografts, Cancer Lett., 110, 149, 1996. 29. Ibuki, Y., Mizuno, S., and Goto, R., γ-Irradiation-induced DNA damage enhances NO production via NF-κB activation in RAW264.7 cells, Biochim. Biophys. Acta, 1593, 159, 2003. 30. Kretzschmar, M., Meisterernst, M., and Roeder, R.G., Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II, Proc. Natl. Acad. Sci. USA, 90, 11508, 1993. 31. Yeh, Y.C. et al., Mammalian topoisomerase I has base mismatch nicking activity, J. Biol. Chem., 269, 15498, 1994. 32. Rossi, F. et al., Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I, Nature, 381, 80, 1996. 33. Rossi, F. et al., The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity, Nucleic Acids Res., 26, 2963, 1998. 34. Castano, I.B. et al., Mitotic chromosome condensation in the rDNA requires TRF4 and DNA topoi- somerase I in Saccharomyces cerevisiae, Genes Dev., 10, 2564, 1996. 35. Walowsky, C. et al., The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin, J. Biol. Chem., 274, 7302, 1999. 36. Stork, G. and Schultz, A.G., The total synthesis of dl-camptothecin, J. Am. Chem. Soc., 93, 4074, 1971. 37. Jew, S.-S. et al., Synthesis and antitumor activity of camptothotecin analogues, Korean J. Med. Chem., 6, 263, 1996. Copyright © 2005 CRC Press, LLC 38. Thomas, C.J., Rahier, N.J., and Hecht, S.M., Camptothecin: current perspectives, Bioorg. Med. Chem., 12, 1585, 2004. 39. Corey, E.J., Crouse, D.N., and Anderson, J.E., A total synthesis of natural 20(S)-camptothecin, J. Org. Chem., 40, 2140, 1975. 40. Wani, M.C., Nicholas, A.W., and Wall, M.E., Plant antitumor agents 28. Resolution of a key tricyclic synthon, 5′(RS)-1,5-dioxo-5′-ethyl-5′-hydroxy-2′H,5′H,6′H-6′-oxopyrano[3′,4′-f]∆6,8-tetrahydro- indolizine: total synthesis and antitumor activity of 20(S)- and 20(R)-camptothecin, J. Med. Chem., 30, 2317, 1987. 41. Terasawa, H. et al., Antitumor agents III. A novel procedure for inversion of the configuration of a tertiary alcohol related to camptothecin, Chem. Pharm. Bull., 37, 3382, 1989. 42. Ejima, A. et al., Antitumour agents. Part 2. Asymmetric synthesis of (S)-camptothecin, J. Chem. Soc., Perkin Trans. 1, 27, 1990. 43. Comins, D.L., Hong, H., and Jianhua, G., Asymmetric synthesis of camptothecin alkaloids: A nine- step synthesis of (S)-camptothecin, Tetrahedron Lett., 35, 5331, 1994. 44. Comins, D.L. and Nolan, J.M., A practical six-step synthesis of (S)-camptothecin, Org. Lett., 3, 4255, 2001. 45. Tagami, K. et al., Asymmetric synthesis of (+)-camptothecin and (+)-7-ethyl-10-methoxycamptothe- cin, Heterocycles, 53, 771, 2000. 46. Bennasar, M.-L. et al., Addition of ester enolates to N-alkyl-2-fluoropyridinium salts: total synthesis of (±)-20-deoxycamptothecin and (+)-camptothecin, J. Org. Chem., 67, 7465, 2002. 47. Ciufolini, M.A. and Roschangar, F., Practical synthesis of (20 S)-(+)-camptothecin: the progenitor of a promising group of anticancer agents, Targets Heterocyclic Syst., 4, 25, 2000. 48. Imura, A., Itoh, M., and Miyadera, A., Enantioselective synthesis of 20(S)-camptothecin using an enzyme-catalyzed resolution, Tetrahedron: Asymmetry, 9, 2285, 1998. 49. Fang, F.G., Xie, S., and Lowery, M.W., Catalytic enantioselective synthesis of 20(S)-camptothecin: a practical application of the Sharpless asymmetric dihydroxylation reaction, J. Org. Chem., 59, 6142, 1994. 50. Jew. S.-S. et al., Enantioselective synthesis of 20(S)-camptothecin using Sharpless catalytic asymmet- ric dihydroxylation, Tetrahedron: Asymmetry, 6, 1245, 1995. 51. Blagg, B.S.J. and Boger, D.L., Total synthesis of (+)-camptothecin, Tetrahedron, 58, 6343, 2002. 52. Curran, D.P. et al., The cascade radical annulation approach to new analogues of camptothecins. Combinatorial synthesis of silatecans and homosilatecans, Ann. N. Y. Acad. Sci., 922, 112, 2000. 53. Yabu, K. et al., Studies toward practical synthesis of (20S)-camptothecin family through catalytic enantioselective cyanosilylation of ketones: improved catalyst efficiency by ligand-tuning, Tetrahedron Lett., 43, 2923, 2002. 54. Sawada, S. et al., Synthesis and antitumor activity of 20(S)-camptothecin derivatives: carbamate- linked, water-soluble derivatives of 7-ethyl-10-hydroxycamptothecin, Chem. Pharm. Bull., 39, 1446, 1991. 55. Kingsbury, W.D. et al., Synthesis of water-soluble (aminoalkyl)camptothecin analogs: inhibition of topoisomerase I and antitumor activity, J. Med. Chem., 34, 98, 1991. 56. Lackey, K. et al., Rigid analogues of camptothecin as DNA topoisomerase I inhibitors, J. Med. Chem., 38, 906, 1995. 57. Ihara, M. et al., Studies on the synthesis of heterocyclic compounds and natural products. 999. Double enamine annelation of 3,4-dihydro-1-methyl-β-carboline and isoquinoline derivatives with 6-methyl- 2-pyrone-3,5-dicarboxylates and its application for the synthesis of (+/-)-camptothecin, J. Org. Chem., 48, 3150, 1983. 58. Kurihara, T. et al., Synthesis of C-nor-4,6-secocamptothecin and related compounds, J. Heterocycl. Chem., 30, 643, 1993. 59. Subrahmanyam, D. et al., Novel C-ring analogues of 20(S)-camptothecin Part 2. Synthesis and in vitro cytotoxicity of 5-C-substituted 20(S)-camptothecin analogues, Bioorg. Med. Chem. Lett., 9, 1633, 1999. 60. Sugimori, M. et al., Antitumor agents. VI. Synthesis and antitumor activity of ring A-, ring B-, ring C-modified derivatives of camptothecin, Heterocycles, 38, 81, 1993. 61. Camptothecins: New Anticancer Agents, Potmesil, M. and Pinedo, H., Eds., CRC Press, Boca Raton, 1995. Copyright © 2005 CRC Press, LLC 62. Kawato, Y. and Terasawa, H., Recent advances in the medicinal chemistry and pharmacology of camptothecin, in Progress in Medicinal Chemistry, Ellis, G.P., and Luscombe, D.K., Eds., Elsevier, London, 1997, Vol 34, pp. 70–100. 63. Wall, M.E. and Wani, M.C., Recent advances in the medicinal chemistry and pharmacology of camptothecin, in Cancer Chemotherapeutic Agents, Foye, W.O., Ed., American Chemical Society, Washington D.C., 1995, pp. 293–310. 64. Wani, M.C. et al., Plant antitumor agents 18. Synthesis and biological activity of camptothecin analogues, J. Med. Chem., 23, 554, 1980. 65. Wani, M.C., Nicholas, A.W., and Wall, M.E., Plant antitumor agents 23. Synthesis and antileukemic activity of camptothecin analogs, J. Med. Chem., 29, 2358, 1986. 66. Wani, M.C. et al., Plant Antitumor Agents 25. Total synthesis and antileukemic activity of ring A substituted camptothecin analogues. Structure-activity correlations, J. Med Chem., 30, 1774, 1987. 67. Wall, M.E. et al., Plant antitumor agents 30. Synthesis and structure activity of novel camptothecin analogues, J. Med. Chem., 36, 2689, 1993. 68. Jaxel, C. et al., Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity, Cancer Res., 49, 1465, 1989. 69. Wang, X., Zhou, X., and Hecht, S.M., Role of the 20-hydroxyl group in camptothecin binding by the topoisomerase I-DNA binary complex, Biochemistry, 38, 4374, 1999. 70. Hertzberg, R.P. et al., Modification of the hydroxy lactone ring of camptothecin: Inhibition of mam- malian topoisomerase I and biological activity, J. Med. Chem., 32, 715, 1989. 71. Burke, T.G. and Mi, Z., Ethyl substitution at the 7 position extends the half-life of 10-hydroxycamp- tothecin in the presence of human serum albumin, J. Med. Chem., 36, 2580, 1993. 72. Lavergne, O. et al., BN80245: An E-ring modified camptothecin with potent antiproliferative and topoisomerase I inhibitory activities, Bioorg. Med. Chem. Lett., 7, 2235, 1997. 73. Cagir, A. et al., Luotonin A. A naturally occurring DNA topoisomerase I poison, J. Am. Chem. Soc., 125, 13628, 2003. 74. Luzzio, M.J. et al., Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I, J. Med. Chem., 38, 395, 1995. 75. van Hattum, A.H. et al., The activity profile of the hexacyclic camptothecin derivative DX-8951f in experimental human colon cancer and ovarian cancer, Biochem. Pharmacol., 64, 1267, 2002. 76. Josien, H. et al., 7-Silylcamptothecins (silatecans): a new family of camptothecin antitumor agents, Bioorg. Med. Chem. Lett., 7, 3189, 1997. 77. Dallavalle, S. et al., Novel 7-oxyiminomethyl derivatives of camptothecin with potent in vitro and in vivo antitumor activity, J. Med. Chem., 44, 3264, 2001. 78. Pollack, I.F. et al., Potent topoisomerase I inhibition by novel silatecans eliminates glioma proliferation in vitro and in vivo, Cancer Res., 59, 4898, 1999. 79. Bom, D. et al., Novel A, B, E-ring-modified camptothecins displaying high lipophilicity and markedly improved human blood stabilities, J. Med. Chem., 42, 3018, 1999. 80. Walker, M. A. et al., Synthesis of an immunoconjugate of camptothecin, Bioorg. Med. Chem. Lett., 12, 217, 2002. 81. Arimondo, P.B. et al., Design and optimization of camptothecin conjugates of triple helix-forming oligonucleotides for sequence-specific DNA cleavage by topoisomerase I, J. Biol. Chem., 277, 3132, 2002. 82. Wang, C.C.C. and Dervan, P.B., Sequence-specific trapping of topoisomerase I by DNA binding polyamide-camptothecin conjugates, J. Am. Chem. Soc., 123, 8657, 2001. 83. Conover, C.D. et al., Camptothecin delivery systems: enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker, Cancer Chemother. Pharmacol., 42, 407, 1998. 84. Greenwald, R.B., Zhao, H., and Xia, J., Tripartate poly(ethylene glycol) prodrugs of the open lactone form of camptothecin, Bioorg. Med. Chem., 11, 2635, 2003. 85. Garcia-Carbonero, R. and Supko, J.G., Current perspectives on the clinical experience, pharmacology, and continued development of the camptothecins, Clinical Cancer Res., 8, 641, 2002. 86. Ulukan, H. and Swaan, P.W., Camptothecins. A review of their chemotherapeutic potential, Drugs, 62, 2039, 2002. Copyright © 2005 CRC Press, LLC 87. Kim, D.-K. and Lee, N., Recent advances in topoisomerase I-targeting agents, camptothecin analogues, Mini Rev. Med. Chem., 2, 611, 2002. 88. Dallavalle, S. et al., Perspectives in camptothecin development, Expert Opin. Ther. Patents, 12, 837, 2002. 89. Bailly, C., Homocamptothecins: Potent topoisomerase I inhibitors and promising anticancer drugs, Critical Rev. Oncology/Hematology, 45, 91, 2003. 90. Hatefi, A. and Amsden, B., Camptothecin delivery methods, Pharm. Res., 19, 1389, 2002. Copyright © 2005 CRC Press, LLC 3 The Discovery and Development of the Combretastatins Kevin G. Pinney, Christopher Jelinek, Klaus Edvardsen, David J. Chaplin, and George R. Pettit CONTENTS I. Introduction, History, and Structures of Combretastatins II. Biochemical and Biological Mechanism of Action III. Therapeutic Intervention through Vascular Targeting IV. Synthesis of Combretastatins V. Combretastatin Derivatives and Synthetic Analogs Inspired by the Combretastatins VI. Preclinical Studies A. Effects of CA4P on Tumor Blood Flow B. Tumor Response to CA4P as a Single Agent C. Treatment Response to CA4P in Combination Therapy D. Preclinical Studies with CA1P (OXi4503) VII. Clinical Experience VIII. Coda IX. Acknowledgments References I. INTRODUCTION, HISTORY, AND STRUCTURES OF COMBRETASTATINS The Combretaceae plant family (comprising 600 or more species) of shrubs and trees is divided among 20 genera, of which the Combretum genus (250 species) of tropical and deciduous trees encompasses the largest number.1 Some 24 species of Combretum are well-known in African folk medicine for applications and problems ranging from heart and worm remedies to wound dressings, treatment for the mentally ill, and scorpion stings.2 Only the Indian Combretum latifolium appears to have been recorded as a folk medical treatment for cancer.3 However, over 30 years ago, as part of the U.S. National Cancer Institute’s (NCI’s) worldwide exploratory survey of terrestrial plants, both Combretum molle4 and Combretum caffrum (Eckl. and Zeyh.) Kuntze were found to provide extracts significantly active against the murine P-388 lymphocytic leukemia (PS system). C. caffrum is a deciduous tree (growing to 15 m high) in Africa, found principally in the Eastern Cape and Transkei to Natal. This willow-like tree (“bushwillow”) is a common sight overhanging stream beds. In autumn, these trees become quite prominent, with displays of reddish-brown fruit and leaves that turn

bright red before falling.5 The powdered root bark has been used by the Zulu of South Africa as a charm to harm an enemy.2 Copyright © 2005 CRC Press, LLC In a broader context, the Combretaceae family is well represented in traditional medical practices of, especially, Africa and India.6 Illustrative is a study of Combretaceae species used in Somalia.7 These species range from Combretum hereroense (young shoots used for respiratory infection) to Terminalia brevipes (root bark employed for hepatitis and malaria) to Commelina forskoolii (juice for treatment of uterine cancer). Nine other species including Anogeissus leiocorpus (fruit and roots as an anthelmintic treatment), Guiera senegalensis (fruit and leaves for leprosy and dysentery), and Quisqualis indica (leaves for vermifuge) are more widely used in Africa.8 Other Combretaceae species, such as C. coccineum and C. constrictum,9 are well-known medicinal plants in Indian Ocean Island areas. Even better known is the medicinal herb C. micronthum, used as an opium antidote for addiction and for other indications from blackwater fever to nausea.10 Importantly, seven species of the genus Terminalia11 and one Guiera species have a long history in African and Asian primitive medical treatments for cancer.3 In Zimbabwe, Combretum molle and three other Combretum species have been summarized along with two Terminalia species as plant extract remedies for diarrhea.12 The early events leading to the discovery of the combretastatins began with the first (1973) collection of C. caffrum in Africa for the NCI in the former Rhodesia (now Zimbabwe). Several scale-up recollections of C. caffrum were completed, and chemical investigations of constituents were pursued by an NCI collaborative research group, but by 1979 that research had led to a dead end in respect to anticancer constituents. In 1979 one of us (G.R.P.) and colleagues continued with a 1979 C. caffrum collection in collaboration with the NCI Natural Products Branch. As summarized in the sequel to this intro- duction, this research subsequently led to the discovery of 20 cancer cell growth inhibitory stilbenes, bibenzyls, dihydrophenanthrenes, and phenanthrenes from C. caffrum. Some of those discoveries led to a number of important advances that range from the first isolation of a cancer cell growth inhibitor, (-)-combretastatin, isolated by using the NCI astrocytoma bioassay, to the first well- established vascular-targeting anticancer drug — sodium combretastatin A-4 phosphate (CA4P), which has entered human cancer clinical trials. Several of the natural products isolated from C. caffrum are characterized by their remarkable biological activity as inhibitors of tubulin polymerization, potent in vitro inhibition against human cancer cell lines, and demonstrated in vivo efficacy as vascular targeting agents (VTAs; in suitable prodrug formulation) and antiangiogenesis agents. The combretastatins are especially noteworthy for their robust activity in biological systems, while maintaining remarkable simplicity in terms of chemical structure. Combretastatin A-4 (CA4) and combretastatin A-1 (CA1), along with their corresponding phosphate prodrug salts (CA4P and CA1P, respectively), have emerged as compounds of profound therapeutic interest (Figure 3.1). Several excellent reviews of the combretastatins and novel analogs inspired by the combretastatins have been published.13–24 In addition, numerous patents have been issued, or are in the application process, for a wide variety of combretastatins and their analogs.25–34 The discovery and development of small-molecule (i.e., compounds with molecular weights in the range of 300–800 g/mol) inhibitors of tubulin assembly has been (and continues to be) a challenging and fruitful goal that, to a large degree, began with colchicine (Figure 3.1) in the 1930s and 1940s. Representative naturally occurring antimitotic ligands19 in addition to colchicine include paclitaxel,35 epothilone A,36 vinblastine,37 dolastatin 10,38,39 and CA4.40–43 In addition, there are a variety of synthetic compounds that also demonstrate efficient inhibition of tubulin polymeriza- tion.42,43 Because of the poor solubility of CA1 and CA4, sodium phosphate prodrugs CA1P44 and CA4P45 were developed in the mid-1990s. Isolation of the first constituent of C. caffrum, (-)-combretastatin (Figure 3.2), as noted above, was achieved in 1982 by Pettit and coworkers.46 By 1987, the absolute stereochemistry, attributed to the single stereogenic center in the molecule, was determined to be the R-configuration.47 The combretastatins are currently divided into four major groups on the basis of their structural char- acteristics. These include the A-Series (cis-stilbenes), B-series (diaryl-ethylenes), C-series (quinone), and D-series (macrocyclic lactones). Representative members of each of these groups are depicted in Figure 3.2. Copyright © 2005 CRC Press, LLC H O 3CO H3CO H3CO N H3CO H3CO O H H 3CO OCH3 OCH OH 3 O Na O P H3CO O Na O OCH3 OCH3 OCH CA4 CA4P Colchicine 3 H3CO H3CO O O Na OH O P H3CO H3CO O Na O OCH3 OCH3 O Na OH O P O Na OCH3 OCH3 CA1 CA1P FIGURE 3.1 Structures of combretastatin A-4, combretastatin A-4 phosphate, combretastatin A-1, combret- astatin A-1 phosphate, and colchicine. The combretastatin A and B series contain two phenyl rings tilted at 50–60˚ to each other, and are linked by a two-carbon bridge.15 The bridge is either unsaturated, as in the case of stilbenes, or saturated. The substituents differ in their respective locations on the phenyl rings, and they usually contain methoxy and hydroxy functionalities. The combretastatin A series is made up of six constituents, combretastatin A-148 (CA1), CA2,49 CA3,49 CA4,50 CA5,50 and CA6,50 which retain a cis-stilbene moiety. The B series contains four compounds — CB1,48 CB2,49 CB3,51 and CB451 — with three similar structures not named according to the prescribed nomenclature.51 These compounds are similar to the A series except for a saturated carbon–carbon bridge between the two aryl rings. There is one compound in the C series, designated combretastatin C152 (CC1), that contains a three-ring quinone structure. Two unusual macrocyclic lactones make up the D series, CD153 and CD2.54 Finally, there are four phenanthrene-like compounds that are currently not named other than by their IUPAC designation.16 II. BIOCHEMICAL AND BIOLOGICAL MECHANISM OF ACTION Antimitotic anticancer drugs, which disrupt the mitotic spindle, have long been a major component in the arsenal of “weaponry” available to wage the battle against cancer. The tubulin–microtubule protein system is the biological target for the majority of these compounds.17,19 Tubulin is a cytoskeletal protein ubiquitous to eukaryotic cells. In addition to providing cells with shape and serving as a cellular mobility (transportation) system, it also plays a key role during mitosis.55 During metaphase of the cell cycle, the tubulin heterodimer (composed of both alpha and beta subunits) undergoes assembly (also referred to as polymerization) to form microtubules, which, collectively, are essential components of the mitotic spindle. The assembly process, which is reversible through a disassembly mechanism, localizes in the microtubule organizing centers (cen- trosomes) of dividing cells and provides a venue by which the microtubules are able to interdigitate between the separating chromosomes, thereby pushing them apart. Certain compounds such as vinblastine, vincristine, colchicine, and podophyllotoxin, function biologically by binding to tubulin heterodimers and causing conformational changes and a steric bias that inhibits the assembly of tubulin into microtubules.17,19 Other compounds, such as paclitaxel and the epothilones, bind to the microtubule itself and inhibit the disassembly process into the tubulin protein.17,19 In both cases, cellular division is disrupted by these compounds, and although healthy cells are also affected, it is the tumor cells that are more prominently targeted because they are characterized by rapid proliferation, which is tubulin dependent. Tubulin is bound by two molecules of guanosine triphosphate (GTP) with a cross-sectional diameter of approximately 24 nm. A tertiary structure of the α,β−tubulin heterodimer (as contained Copyright © 2005 CRC Press, LLC OH H3CO H3CO O HO OH H3CO H3CO O H3CO OCH 3 OCH3 OCH OCH OH OH 3 OH 3 OH OCH3 OCH 3 OCH3 OCH 3 (-) -Combretastatin CA1 CA2 CA3 OCH3 H3CO HO OCH3 H O H O HO 3C 3C OCH3 OCH OH 3 OCH3 H3CO OCH3 OCH3 OCH 3 CA4 CA5 CA6 (synthetic analog) H3CO HO H O 3CO H3C OH H H 3CO H3CO 3CO OCH3 OCH OH OCH3 3 OCH3 OH OH OH OCH OH 3 OCH OH 3 CB1 CB2 CB3 CB4 H CO H 3 3CO H3CO H3CO OCH3 OCH3 OCH OH 3 OH OCH3 OH Not Named Not Named Not Named O OH OH H3CO O O H3CO O O OH O O OCH 3 O O CC1 CD1 CD2 H3CO H HO 3CO H3CO H3CO H CO H 3 3CO H3CO OCH3 OCH3 OCH3 OH OCH3 OH OH OH OCH3 OCH CH 3 OH O 3 Not Named Not Named Not Named Not Named FIGURE 3.2 Natural products isolated from Combretum caffrum. in the microtubule stabilized by taxol) was reported in 1998 by Downing and coworkers at a resolution of 3.7 Å, using electron crystallography.56 This accomplishment marks the culmination of decades of work directed toward the elucidation of this structure and should contribute to the identification of small-molecule binding sites, such as the colchicine site, through techniques such as photoaffinity and chemical affinity labeling.57–70 Small molecules can bind in at least three known locations or sites on the tubulin system: the colchicine site, the vinca-alkaloid site, and the taxoid site. These sites are named because of the binding of colchicine, vinblastine, and taxol, respectively, to certain distinct binding pockets on the tubulin dimer itself, or on the microtubule in the case of taxol.71 Ligands that interact at the taxoid site (such as paclitaxel and the epothilones) stabilize the microtubule, whereas ligands interacting at the vinca domain (such as vinblastine) or the colchicine Copyright © 2005 CRC Press, LLC site (such as colchicine and CA4) disrupt the formation of microtubules. The combretastatin A series is historically known for its remarkable biological activity in terms of inhibition of tubulin assembly and in vitro cytotoxicity against human cancer cell lines. Combretastatin A1 and CA4 are among the most active of the natural products isolated from C. caffrum in terms of in vitro activity16 (Table 3.1). III. THERAPEUTIC INTERVENTION THROUGH VASCULAR TARGETING Although antimitotic agents attack the tumor cells directly and disrupt mitosis, some molecules have been shown to exhibit an antivascular and antiangiogenic effect as well. Often, the two classes overlap, as in the case of the combretastatins, where certain compounds that cause direct vascular damage can also prevent angiogenesis. Vascular targeting agents (VTAs) function by destroying existing tumor vasculature through a mechanism that induces morphological changes within endot- helial cells, thereby disrupting shape, transport, and motility. In contrast, antiangiogenic agents inhibit the formation of new blood vessels. In either case, this disruption of vasculature essentially starves the tumor. Recently, it has been discovered that certain of these tubulin binding compounds, in prodrug form, demonstrate remarkable selectivity for the destruction of tumor vasculature.72–75 Solid tumor survival is dependent on blood, oxygen, and nutrient supply from surrounding vessels.76 Growing tumors receive their nutrients through existing vasculature in the surrounding area as well as through newly formed neovascularization (angiogenesis). In prodrug form, these VTAs are, for the most part, protected from binding to tubulin. However, once they undergo enzymatic cleavage to their corresponding parent free phenol or amine analog, they function as potent inhibitors of tubulin assembly. In the microvessels feeding tumor cells, these compounds bind to endothelial cell tubulin and cause rapid morphology changes in the endothelial cells lining the microvessels.77–79 These morphology changes (known as rounding up of the cells) disrupt blood flow, which ultimately renders both new microvessels as well as mature and established vessels nonfunctional for blood delivery, causing hypoxia and necrosis of the tumor. The reason for the selectivity of the VTAs for the microvessels of tumors is largely unknown. However, it may reflect, in part, variability in the cytoskeletal make-up of rapidly proliferating endothelial cells inherent to microvessels feeding tumor cells versus the normal proliferating endothelial cells of microvessels serving healthy cells.77 Because one single microvessel can feed hundreds or thousands of tumor cells, this method of vascular targeting has the potential to be extremely effective in destroying tumor cells without targeting them directly (as it is the microvessels that are immediately affected). CA4P and other VTAs are useful as chemotherapeutic agents for cancer treatment as well as for therapeutic intervention in ocular diseases (such as wet, age-related macular degeneration).80 At present, there are at least three compounds in human clinical development as potential VTAs for cancer chemotherapy that use a tubulin-based biological mechanism. Although none of these compounds has achieved NDA (New Drug Application) approval for marketing by

the U.S. Food and Drug Administration, CA4P is the most advanced in clinical trials. In June 2003, CA4P received orphan drug approval by the U.S. Food and Drug Administration for the thyroid cancer group, and a month later it received approval for a “fast-track” phase II clinical trial against the usually quite lethal (median survival time is 4 months) anaplastic thyroid cancer. IV. SYNTHESIS OF COMBRETASTATINS Isolation of CA1 and CA4, as with the other constituents, began with a methylene chloride-methanol extract, initially from the macerated branches, leaves, and fruit, and subsequently from the stem wood of C. caffrum.48,50,81 These extracts were then separated by bioassay (PS system) guided techniques. For example, with respect to CA4, a trace fraction was isolated from 77 kg of dry stem wood. Further separation led to 26.4 mg of a fraction that at first seemed to be a pure compound, but high-field 1H Copyright © 2005 CRC Press, LLC TABLE 3.1 In Vitro Activities of Combretastatins Name Structure Tubulina P388b L1210c Name Structure Tubulina P388b L1210c OH 5–7.5 0.011d 0.05 CB4 H3CO OH 25–30 1.7h 1 H3CO H3CO H3CO OCH3 OH OCH3 OCH3 OCH3 CA1 H3CO 2–3 0.99e 0.6 — O 10–15 NA 1 H3CO OH H3CO OCH3 H3CO OH O OCH3 OH OCH3 CA2 O 4–5 0.027f 0.1 — OH >100 NA 4 O O OCH3 O OH OCH3 O O CA3 HO 4–5 0.026f 0.04 — OH >100 NA 50 O H3CO OCH3 O OH OCH3 O Copyright © 2005 CRC Press, LLC CA4 1.9g 0.0034 0.007 CC1 NA 2.2i NA H H 3CO 3CO 2–3 H3CO OCH OCH3 3 OH OH OH OCH3 trans H 7.5–10 0.050 0.02 CD1 NA 3.3j NA 3CO H3CO CA4 OH H3CO OCH3 OCH3 OH OH OCH3 OCH3 CA5 H3CO 75–100 0.9 2 CD2 HO NA 5.2k 51l H3CO OCH3 OCH3 OH OH OCH3 CA6 H3CO >100 18 30 — H3CO 100 NA 1 H3CO H3CO OCH3 OCH3 OH OH OH CB1 H3CO 3e 1.7e 2 — H3CO >100 NA 3 4–5 H3CO H3CO OCH3 OH OCH3 OH OCH3 OCH3 Copyright © 2005 CRC Press, LLC TABLE 3.1 In Vitro Activities of Combretastatins Name Structure Tubulina P388b L1210c Name Structure Tubulina P388b L1210c CB2 3 40 0.32f H3CO OCH 3 — H3CO >100 NA 20 H3CO H3CO OCH3 OH OH HO OCH3 CB3 HO >100 0.4h 3 — HO >100 NA 20 H3CO H3CO OCH3 OCH OCH 3 3 OH OCH3 OCH3 Note: The compound is (-)-combretastatin. a Indicates tubulin polymerization inhibition (IC50 micromolar) (data taken from ref. 16, except as noted). b Indicates cytotoxicity for the PS cell line (P388 leukemia) (ED50 in µg/mL) (data taken from ref. 50, except as noted). c Indicates cytotoxicity for the L1210 cell line (murine leukemia) (ED50 micromolar) (data taken from ref. 16, except as noted). d Data taken from ref. 46. e Data taken from ref. 48. f Data taken from ref. 49. g Data taken from ref.13. h Data taken from ref.51. i Data taken from ref.52. j Data taken from ref. 53. k Data taken from ref. 54. l Data taken from ref. 83. Copyright © 2005 CRC Press, LLC OH CHO CHO DIPEA, TBSCl NaBH4 OH DMF OTBS EtOH OTBS 95% 85% OCH3 OCH3 OCH3 H3CO CHO Br H3CO Br PPh3 OCH3 OTBS H3CO OCH3 PBr3 PPh OCH Z 3 3 OCH3 + CH2Cl2 OTBS Toluene n-BuLi, THF 65% 77% OTBS OCH3 OCH3 OCH3 Z/E: 1:1.5 93% total yield H3CO OTBS H3CO H3CO H3CO OCH TBAF 3 H3CO E H3CO THF OCH 93% OCH3 3 OH OTBS OCH3 OCH3 CA4 H3CO H3CO CCl4, DIPEA, DMAP i) NaI, TMSCl (BnO)2P(O)H CH3CN H3CO H O 3CO CH ii) NaOCH O 3CN OCH3 OBn 3, CH3OH OCH Na 98% O P 90% 3 O OBn O P O Na OCH3 OCH3 CA4P SCHEME 3.1 Synthesis of combretastatin A-4 and its disodium phosphate prodrug. and 13C-NMR (nuclear magnetic resonance) indicated a mixture of three compounds. Conversion to a t-butyldimethylsilyl ether derivative and further separation afforded about 9 mg of CA4 silyl ether. Following cleavage of the protecting group from a 1.7-mg sample, a 1.15-mg sample of pure CA4 was obtained. By analogous techniques, CA5 and CA6 were obtained as the other two components of this complex mixture. The structure and configuration of CA4 was established by 1H-NMR spectra,50 which gave evidence of four methoxy groups and seven protons in the aromatic region. Two of the methoxy groups were equivalent, and three protons were displayed at δ 6.734 (d, J = 8.4 Hz), 6.80 (dd, J = 8.42, 2.04 Hz), and 6.92 (d, J = 2.04 Hz), indicating ortho–ortho and ortho–meta coupling, and one singlet at δ 6.53 represented the two symmetrical aromatic protons on the A ring of CA4. The cis (Z) stereochemistry of the stilbene was indicated by the coupling constant (J = 12.16 Hz) for each of the two doublets at d 6.471 and d 6.412. The trans(E) derivative of CA4 showed a vinyl coupling of J = 16.30 Hz. The synthesis of CA4,50 (Scheme 3.1) was achieved through a Wittig reaction between the silyl-protected phosphonium bromide from isovanillin and 3,4,5-trimethoxybenzaldehyde. The Z geometry was confirmed by an x-ray crystal structure determination. The Z isomer was then desilylated with tetrabutylammonium fluoride to afford CA4. Similarly, the structure and configuration of CA148 was confirmed to be Z with an AB system of doublets at δ 6.453 (J = 12.2 Hz) and 6.523 (J = 12.2 Hz) and a two-proton signal at δ 5.438, representing the presence of phenolic groups. The synthesis of CA148 (Scheme 3.2) was also achieved through a Wittig reaction; however, availability and inefficiency of certain starting mate- rials required that 2,3,4-trihydroxybenzaldehyde be used as a starting substance. After selective methylation at the para position and silylation, the benzaldehyde was allowed to react with the phosphonium bromide of 3,4,5-trimethoxybenzyl alcohol to afford the protected Z isomer. After deprotection using tetrabutylammonium fluoride, CA1 was obtained. Copyright © 2005 CRC Press, LLC CHO CHO CHO H3CO OH OH OTBS Borax, NaOH DIPEA, TBSCl OTBS H3CO OH H2O, Me2SO4 DMF OH OTBS 72% 87% OCH3 OTBS OH OCH OCH 3 3 nBuLi OCH3 Br Z OH Br THF PPh3 Z/E: 9:1 + PBr3 PPh3 93% total yield OCH3 H C 3CO OCH H2Cl2 3 H3CO OCH Toluene 3 93% OCH H3CO OCH3 H3CO 3 OCH3 OTBS OCH3 OTBS H3CO OCH3 E H3CO H3CO TBAF (BnO)2P(O)H OH OTBS H3CO H3CO THF CCl , DIPEA, DMAP, CH3CN 68% OCH 4 OCH 3 3 OH 97% OTBS OCH OCH 3 3 CA1 H3CO H3CO O O Na OBn O i) NaI, TMSBr O P O P H3CO OBn CH3CN H3CO O Na O O OCH OCH Na 3 OBn 3 O O P ii) NaOCH3 O P OBn CH3OH O Na OCH OCH3 3 81% CA1P SCHEME 3.2 Synthesis of combretastatin A-1 and its prodrug combretastatin A-1 phosphate. The sodium phosphate salt of CA482 and disodium phosphate salt of CA144 were both synthe- sized via an efficient sequence using a dibenzylphosphite-carbon tetrachloride procedure, followed by debenzylation with sodium iodide and chlorotrimethylsilane. In addition to the Wittig reaction used by Pettit and coworkers, other methods have been developed to synthesize CA4 and CA1, including a Perkin condensation of 3,4,5-trimethoxyphe- nylacetic acid and 3-hydroxy-4-methoxybenzaldehyde,85 a Suzuki cross-coupling of an ethenyl bromide and 3,4,5-trimethoxyphenyl boronic acid,85 a biooxidation of arenes to catechols using Escherichia coli JM109 followed by a Pd-catalyzed Suzuki coupling,86 and a modified Suzuki reaction through a hydroboration/protonation of a diaryl-alkyne.87 Further study of C. caffrum constituents led to the isolation, characterization, and synthesis of B, C, and D series members. In general, B series constituents were synthesized by methods analogous to A series syntheses followed by hydrogenation.48,49,51 For example, for CB1,48 a mixture of CA1 in methanol and 5% Pd/C was treated with a positive pressure of H2 at ambient temperature overnight to afford CB1. Synthesis of CC152 (Scheme 3.3) was achieved through photochemical cyclization of the cor- responding stilbene. The improved synthesis of CC1 included selective demethylation of 2,3,4- trimethoxybenzaldehyde with aluminum chloride followed by silylation. Subsequent coupling of the aldehyde with the corresponding ylide and photochemical cyclization, using iodine and a mercury lamp, provided the phenanthrene product. Desilylation using tetrabutylammonium fluoride afforded the phenol, which, on Fremy’s salt oxidation, yielded the quinone CC1. The unusual macrocyclic lactones, CD1 and CD2, have been synthesized by several groups.83,88–90 Boger’s synthesis using an intramolecular Ullmann macrocyclization83 to afford CD2 is presented here (Scheme 3.4). Esterification of 3-(3-hydroxy-4-methoxyphenyl)propanoic acid with (Z)-3-(4-iodophenyl)-2-propenol under Mitsunobu conditions provided the bridged diaryl ester. Copyright © 2005 CRC Press, LLC OH CHO CHO DIPEA, TBSCl NaBH4 OH DMF OTBS 95% EtOH OTBS OCH OCH 85% 3 3 OCH3 Br TBSO Br PPh H3CO 3 PBr3 PPh3 H3CO CH2Cl2 OTBS Toluene 65% 77% OTBS OTBS OCH3 OCH3 nBuLi Z OCH3 THF CHO CHO CHO + Z/E: 1:2.5 OTBS OCH3 OH AlCl TBSCl OTBS 98% total OCH yield OTBS 3 3 OCH3 Benzene OCH DIPEA H3CO 91% 3 DMF OCH3 OCH3 OCH3 98% OCH3 H3CO E TBSO TBSO H3CO H I 3CO 2 , hv H3CO benzene-hexanes H3CO 49% OTBS OTBS (Z+E mixture) OCH3 (after separation) OCH3 HO O H CO H 3 3CO TBAF Fremy's salt H H3CO T 3CO HF CH3OH-H2O O OH 54% OH OCH3 OCH3 CC1 SCHEME 3.3 Synthesis of combretastatin C1. Upon intramolecular Ullman macrocyclization with CuCH3 and pyridine, followed by aryl methyl ether deprotection, CD2 was obtained. V. COMBRETASTATIN DERIVATIVES AND SYNTHETIC ANALOGS INSPIRED BY THE COMBRETASTATINS Many compounds (Figs. 3.3 and 3.4) have been synthesized to mimic the structure and efficacy of the combretastatins, especially CA4 and CA1.13,16,91 A wide variety of combretastatin analogs with functional group modification have been prepared with the A and B aryl rings and the ethylene bridge intact. A sampling of these compounds includes 3-nitro,64,92 3-amino,64,92 3-amino acid salts,92,93 2- nitrogen substituted derivatives,94 3-azido,64 3,4-methylenedioxy-3-amino derivatives,95 3-fluoro,96 4- methyl,97 and 3,4,5-trimethyl98 combretastatin analogs (Figure 3.3). In addition, many combretastatin analogs feature significant structural variation from the basic stilbenoid molecular core structure. A representative group (not intended to be inclusive) includes phenstatin,99 hydroxyphenstatin,100 het- erocombretastatins,101 combretatropones,102,103 aza-combretastatins,104 bridge-modified vicinal diols,105,106 combretadioxolane analogs,107–109 chalcone derivatives,110 disubstituted imidazole ana- logs,111 pyridone analogs,112 and sulfonamide analogs113,114 (Figure 3.3). In addition, 3-aroyl-2-aryl indoles,72,115,116 2,3-diaryl indoles,116 3-formyl indoles,117 benzo[b]thiophene,43,118-123 and benzofuran analogs,116,124,125 along with dihydronaphthalenes,119,126,127 dihydroxyindolo[2,1-α]isoquinolines,128 Copyright © 2005 CRC Press, LLC CHO CO2CH3 (CF3CH2O)2P(O)CH2CO2CH3 DIBAL-H 18-crown-6, KHMDS 80% 97% I I CO2H OH OCH3 I HO OH OCH CuCH 3 3 PPH3 O pyridine/reflux DEAD I 37% 97% O OCH3 OH O BI O 3 dimethylaniline 37% O O O O CD2 SCHEME 3.4 Synthesis of CD2. and indolyloxazoline analogs,129,130 have been prepared (Figure 3.4). The relative simplicity of the basic stilbenoid molecular architecture characteristic of CA4 and CA1, along with the ease of synthesis and the versatility afforded by the variety of synthetic routes available for the formation of stilbenoids, has resulted in extensive structure activity relationship studies around the combretastatins. These studies have led to the discovery and development of a large number of structurally distinct combre- tastatin analogs. In fact, the literature, with regard to the combretastatins, has exploded in the last 10 years. For example, in 1994, a literature search for CA4 would turn up approximately 30 “hits,” and a search today (2004) yields over 600 papers and professional presentations. Three small-molecule VTAs that are currently in human clinical development function through a biological mechanism ultimately involving the tubulin–protein system (Figure 3.5). These drugs include CA4P (discovered by George R. Pettit, Arizona State University, sponsored by Oxigene, Inc.; see appropriate references throughout), AVE806292,93,131 (discovered by Ajinomoto, Inc. [AC7700] and sponsored by Aventis), and ZD-6126132–135 (licensed from Angiogene Pharmaceuti- cals UK and sponsored by AstraZeneca). The similarity of CA4P and AVE8062 to the natural product CA4 is obvious. Likewise, the six, seven, six ring fusion in ZD-6126 is highly reminiscent of the six, seven, seven (tropolone) ring system of the natural product colchicine. VI. PRECLINICAL STUDIES The recent interest in the anticancer potential of the combretastatins has gained momentum from the observation that CA4P can induce rapid and selective blood flow shutdown within tumor tissue.76,136,137 As the development and activity of CA4P as a VTA has been the subject of several recent reviews15,22,74,138 only a brief summary of the

preclinical data will be provided here. A. EFFECTS OF CA4P ON TUMOR BLOOD FLOW A number of studies have demonstrated the rapid and selective effects of CA4P on tumor vascular function.41,137,139–148 These studies have established that blood flow reductions occur in transplanted and spontaneous rodent tumors and in xenografted human tumors. Blood flow effects can be measured within 10–20 minutes of CA4P administration, a timeframe that closely mimics the in vitro effects on endothelial cell shape. These blood flow effects persist for hours, although in some Copyright © 2005 CRC Press, LLC H3CO O H3CO H3CO O H3CO CH3O CH3O CH3O R R F OCH3 OCH3 OCH3 R = NO2 R = NH2, NH3Cl, NH-Cys R = NH2 (AC-7739, AVE8063A) (and other amino acid derivatives) 3'-Fluoro-combretastatin R = NH-serinamide salt (AC-7700, AVE 8062A) R = N3 O O OH H3CO H3CO OH H3CO OH H3CO H3CO OCH3 H3CO OCH3 CH3O CH3O CH3O R CH3 R = OH, O-Phosphate Salt Phenstatin Hydroxyphenstatin 4'-Methylcombretastatin Analog O H3CO H3CO H3CO N H H 3CO N H 3CO 3CO CH CH 3O CH 3O 3O O OH OCH OCH 3 3 Heterocombretastatin Analog Combretatropone Analog Aza-combretastatin Analog OH O O H3CO OH H3CO O H3CO R R H3CO H3CO H3CO CH3O CH3O CH3O OH R OCH3 OCH3 OCH3 OH R = H, OH R = H, OH Bridge-modified Vicinal Diols Combretadioxolane Analogs Chalcone Derivative H3CO OCH3 F H3CO F F F OCH3 N H N N H3CO F S OCH3 CH F O O 3O N O N CH CH3 3 1,2-Disubstituted Imidazole Analog Pyridone Analog Sulfonamide Analog (T138067, T67) FIGURE 3.3 Combretastatin derivatives. tumors restoration of flow can be observed 24 hours following treatment. Because it is known that dramatic tumor cell kill is induced within a few hours of cutting off the blood supply,149 even short periods of blood flow reduction are expected to induce a significant reduction in tumor cell burden. B. TUMOR RESPONSE TO CA4P AS A SINGLE AGENT Measurements of clonogenic tumor cell survival in tumors excised following CA4P treatment have shown that extensive tumor cell kill is induced.41,150 Indeed, these studies show that 24 hours after Copyright © 2005 CRC Press, LLC OCH OCH3 3 OCH OCH3 3 O O Y H OCH OCH3 3 OCH OCH3 OCH 3 3 H N 3CO H S 3CO H N 3CO H H OH R R = H, OH Y = carbonyl, O 3-Aroyl-2-aryl-indole-based Analog 3-Aroyl-2-aryl-benzo[b]thiophene Analogs 3-Formylindole Analog OCH3 OCH H 3 3CO H3CO OCH3 OCH3 H3CO OCH3 O OCH3 OCH3 N H N H 3CO 3C N H3CO H OH OH 2,3-Diaryl indole Analog Indolyloxazoline Analog (A-289099) Dihydronaphthalene-based Analog HO C4H9 Rn N Y OH O H O 3CO H Rn Isoquinoline Analog Y = carbonyl, covalent bond Rn = OCH3, OH Benzofuran Analogs FIGURE 3.4 Synthetic analogs inspired by the combretastatins. a dose of 100 mg/kg, tumor cell survival is reduced by 90%–99% in a number of experimental tumors. Despite the extensive cell kill observed following the vascular shutdown achieved with CA4P, significant growth retardation is very rarely seen. This lack of retardation has been attributed to rapid regrowth from the rim of viable cells surviving at the tumor periphery.41 Of interest is that treatment with conventional cytotoxic chemotherapy or radiation at doses that induce similar or O H3CO H3CO H3CO N H H3CO H3CO O H3CO O CH3O O Na CH3O H3CO O P N OH O O Na H O Na OCH O P 3 OCH3 NH2 HCl O Na CA4P CA4 Serinamide ZD-6126 Pettit-ASU Ajinomoto AstraZeneca Oxigene, Inc. Aventis O H H3CO 3CO Natural Products of Origin N H H3CO H3CO Potent inhibitors of tubulin assembly CH3O H3CO OH O OCH3 OCH3 Combretastatin A4 (CA4) Colchicine {natural product} {natural product} Pettit -- ASU FIGURE 3.5 Small-molecule vascular targeting agents in clinical development. Copyright © 2005 CRC Press, LLC lower levels of cell kill can induce significant retardation of tumor growth.74 One possible expla- nation for this difference is that the cells in the periphery have access to a more nutritive blood flow than those in the center, which are dependent on the relatively poor and chaotic vascular network present within the tumor mass. This has several consequences, one of which is that the hypoxic and nutrient-deprived cells that are more prevalent in the tumor core will be more resistant to conventional treatments such as radiation and cytotoxic approaches, which are known to be more toxic to well-oxygenated and rapidly proliferating cell populations.151 Therefore, if this is the case, treatments such as radiation and cisplatin, in contrast to CA4P, will preferentially kill cells in the periphery and spare those in the center of the tumor. Thus, if the cells that have survived treatment are already proliferating and adjacent to a good supply of oxygen and nutrients, they will initiate rapid regrowth, whereas, if the surviving cells are less well supplied, there will be a lag phase before proliferation is initiated. If this hypothesis is correct, CA4P, along with other VTAs, offers great potential to enhance the effectiveness of current treatment approaches by the complementary cell killing of different cell populations. C. TREATMENT RESPONSE TO CA4P IN COMBINATION THERAPY Several studies have already indicated the therapeutic potential of combining CA4P with radiation and a variety of chemotherapy agents.41,142,152 In addition, CA4P has been shown to enhance other therapeutic approaches such as hyperthermia, antibody-based approaches, and even antiangio- genic agents.146,153–155 In a study by Siemann and colleagues, the tumor cell kill induced by cisplatin or cyclophos- phamide was enhanced by a factor of 10–500 by post-treatment administration of CA4P.150 Also, no increase in the bone marrow stem-cell toxicity associated with these anticancer drugs was observed, giving rise to a real therapeutic gain. Numerous other studies have shown significant enhancement of the effects of conventional chemotherapeutic agents on tumor response, including cisplatin,41 fluorouracil,156 doxorubicin,157 chlorambucil,15 melphalan,15 CPT-11,158 and Taxol®.41 It has been shown that CA4P, when administered shortly following radiation treatment, can significantly enhance tumor response.77,142,153,154,159 In contrast, no enhancement of radiation damage has been observed in normal tissue.160 Combining VTAs with hyperthermia is another potentially useful approach. Clear evidence exists that blood flow to the tumor is important in determining the response to heat.161 Furthermore, a number of studies have now shown that decreasing tumor blood flow by CA4P treatment can provide an effective means of improving tumor response to hyperthermia.154,162 One of the most dramatic enhancements of activity by CA4P in preclinical models was reported by Pedley and colleagues.155 In a study evaluating the efficacy of a radiolabeled anti-CEA antibody against an established human colon cancer in mice, the researchers found that administration of CA4P could improve long-term tumor control from 0% to 85%. D. PRECLINICAL STUDIES WITH CA1P (OXI04503) The preclinical activity of CA1P has only recently been evaluated.163–166 However, the data show that not only is it a much more potent agent than CA4P, but it can also induce tumor growth delays and regressions when used as a single agent (something not usually observed with CA4P). This enhanced activity was unexpected based on the in vitro data for tubulin binding and for inhibition of cell proliferation for the active parent drug, CA1. Because regressions are observed, it indicates that CA1P, in addition to the vascular effects, is also directly attacking the remaining viable cells at the rim of the tumor mass. One possible explanation for this unexpected activity is that it is metabolized in vivo to a reactive and cytotoxic o-quinone. Supportive evidence for this has been recently provided in a paper by Kirwan and colleagues, who show that CA1 is metabolized by tumor tissue to an agent that covalently binds to the cellular contents of the tumor.167 Copyright © 2005 CRC Press, LLC TABLE 3.2 Completed and Ongoing Clinical Trials with Combretastatin A-4 Phosphate (OXi 2021) in Oncology Clinical Compound Indication/Use Phase Notes Combretastatin A-4P Solid tumors; single agent Phase I Three trials completed Combretastatin A-4P Solid tumors; combination with carboplatin Phase Ib Patient enrollment complete Combretastatin A-4P Head and neck/lung/prostate; combination with Phase I/II Ongoing radiation Combretastatin A-4P Anaplastic thyroid cancer Phase II Ongoing Combretastatin A-4P Anaplastic thyroid cancer in combination with Phase I/II Ongoing chemotherapy and radiotherapy Combretastatin A-4P Advanced ovarian cancer; combination with Phase I/II Ongoing carboplatin and paclitaxel Combretastatin A-4P Advanced colorectal cancer, combination with Phase I/II Ongoing iodine-labeled antibody A5B7 VII. CLINICAL EXPERIENCE80 Under the sponsorship of Oxigene, Inc., CA4P and CA1P have moved into clinical trials. To date, there have been nine clinical trials involving CA4P: three completed and six ongoing (Table 3.2), and aspects of the clinical experience with CA4P have been published by Young and Chaplin.80 Ninety-six patients with advanced malignancies were enrolled in three phase I clinical trials, beginning in 1998. These trials explored the safety and pharmacokinetics of three different intra- venous administration schedules of CA4P as a single agent (i.e., single dose schedule every 21 d [“1x/21”] study); weekly for three consecutive weeks every 28 d (“3x/28” study); and daily for five consecutive days every 21 d (“dx5” study).168–172 Doses as high as 114 mg/m2 (weekly schedule) have been studied. Dose-limiting toxicities have been identified in all three trials, resulting in a maximum tolerated dose in the range of 60–68 mg/m2. Dose-limiting toxicities have included dyspnea, myocardial ischemia, reversible neurological events, and tumor pain. A major objective of these studies, in addition to establishing the maximum tolerated dose, was to evaluate the effects on tumor blood flow. Significant reductions in tumor blood flow were seen in all three clinical trials.168–172 Blood flow reductions were observed at all dose levels evaluated between 52 and 114 mg/m2, with no clear dose response being discernable. Additional studies have shown blood flow reductions down to 30 mg/m2 (Oxigene, Inc., unpublished data). Thus, the data on humans confirm the results in animals that tumor blood flow reductions can be seen at doses at or below the maximum tolerated dose. These findings have provided the impetus to move CA4P into later-stage clinical trials. The preclinical data to date for CA4P indicate that although the compound elicits significant reductions in blood flow, resulting in histologically demonstrable tumor necrosis, it appears to leave a rim of viable tumor cells at the periphery. Replication and growth of these few remaining tumor cells results in an insignificant tumor growth delay in a variety of preclinical models. Because of this phenomenon, the clinical focus was directed at combining CA4P with other treatment therapies and modalities that are more effective at the outer tumor regions. Therefore, the current clinical development of CA4P is mainly focused on combinations with several common chemo- therapeutics agents, with fractionated external beam radiotherapy, and with an antibody-based radioimmunotherapeutic agent. A phase Ib, open-label, single-center, dose-escalation clinical trial of CA4P in combination with carboplatin has recently been completed in cancer patients with nonhematological malignancies that Copyright © 2005 CRC Press, LLC have progressed on standard therapy or for whom no life-prolonging treatment is known. The rationale for combining CA4P and carboplatin stems from their different and potentially comple- mentary mechanisms of action and their nonoverlapping toxicity profiles. Additional active clinical trials include a (phase II) single-agent study to evaluate the safety and efficacy of CA4P in anaplastic thyroid carcinoma on a weekly administration schedule as a single agent. Although clinical effectiveness of CA4P as a single agent was not anticipated, an anaplastic thyroid carcinoma patient experienced a complete pathological response with CA4P alone, and thus, a clinical trial to evaluate the potential of CA4P in this aggressive disease was initiated. Another (phase I/II) clinical trial is also ongoing to evaluate the safety of CA4P in combination with radiation therapy in a variety of solid tumor types, including head and neck, prostate, and non–small cell lung cancer. The fourth active trial is a (phase I/II) safety and tolerability trial of CA4P in combination with carboplatin and paclitaxel in advanced ovarian cancer. A fifth study (phase I/II) recently initiated is CA4P in combination with the 131I-radiolabeled anti-CEA antibody A5B7 for the treatment of colorectal carcinoma. A sixth study (phase I/II) soon to be initiated is CA4P in combination with doxorubicin, cisplatin, and radiotherapy for the treatment of newly diagnosed anaplastic thyroid carcinoma. Combretastatin A-1P (Oxi-4503) has also shown excellent activity in preclinical studies. Oxi- 4503 appears to be able to

cause tumor regressions with single-agent activity, whereas CA4P’s in vivo activity is best with combination studies (i.e., phase Ib with carboplatin). In addition, CA1P has the ability to destroy vessels in all regions of the tumor rather than just at the periphery of the tumor, as is the case with CA4P.166 VIII. CODA In the classic 1962 compilation and discussion of “Medicinal and Poisonous Plants of Southern and Eastern Africa,” Watt and Breyer-Brandwijk described the root bark of C. caffrum (aka salic- ifolium E. Mey.) as the Zulu charm for harming an enemy. “Some of the powdered material is heated with water in a piece of broken earthenware pot, and the points of two assegais are dipped into the boiling solution. The assegai points are brought near the mouth and the operator licks them and spits in the direction of his enemy whose name is shouted out. This is repeated several times, the weapons being thrust in the same direction and finally into the ground.” Commencing with the discovery of the combretastatins, the question has arisen many times as to whether or not some of the indigenous people of Africa and elsewhere had used extracts of C. caffrum for medicinal purposes that included treating cancer. The scientific/medical literature appears to be silent on that issue. In that respect, one of us (GRP) was pleased to be contacted in November 1999 by a South African chemist, Case van Hattem. In the helpful correspondence that ensued, van Hattem provided some interesting information. He reports that, “I have spoken with the chairman of the Transkei Traditional Healers Association, and he confirms that they, ‘the Xhosa people,’ have been using C. caffrum for decades and longer for the treatment of cancer amongst other ailments.” The chairman also cautioned that extracts of C. caffrum should be used in combination with other plants to be effective, presumably to moderate the toxicity. Another useful contribution made by van Hattem was the observation that, “The tribe in the area of C. caffrum are Xhosa and not Zulu. There is a gradual change from C. caffrum in the Xhosa area to C. erythrophyllum in the Zulu area as one travels northwards from East London through the Transkei.” Furthermore, the Xhosa and Zulu do not differentiate between these two botanical species, and in his discussions with Professor A. E. van Wyk, curator of the Herbarium, Department of Botany, University of Pretoria, the conclusion is that C. caffrum and C. erythrophyllum are nearly identical. Until Mr. Hattem’s communications in 1999, none of us involved in the sampling of C. caffrum over 30 years ago, and subsequently, in discovery of the combretastatins, had any knowledge of this plant being used in traditional medical treatments for cancer. From a humanitarian viewpoint, this historical uncertainty is essentially moot. This writer (GRP) thinks the real benefit to the people Copyright © 2005 CRC Press, LLC of Zimbabwe and South Africa will be derived from the eventual worldwide success of the CA4 and CA1 prodrugs in treating human cancer. Those drugs should become two of the least expensive anticancer drugs of all time, greatly benefiting both African countries and their citizens. Indeed, if it were not for the NCI and Arizona State University’s Cancer Research Institute, the discovery of CA4 prodrug and its development to the present phase I and II human cancer clinical trials might have been delayed many decades or centuries into the future. Hence, there needs to be a worldwide realization that it is in everyone’s best interest if the collection of plants and animals for medicinal purposes is facilitated by clear and transparent bioprospecting laws. Both the source country and patients worldwide will then benefit from the investment by governments, universities, and the private sector in the discovery and development of new anticancer and other drugs. IX. ACKNOWLEDGMENTS We extend our appreciation and special thanks to each of the colleagues named as coauthors on publications involving the discovery and development of the combretastatins. In addition, we express appreciation for helpful communications with Professor A. E. van Wyk and Mr. Case van Hattem. REFERENCES 1. Willis, J.C. and Arey Shaw, H.K., A Dictionary of the Flowering Plants & Ferns, 8th ed., Cambridge University Press, London, 1973. 2. Watt, J.M. and Breyer-Brandwijk, M.G., The Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd ed., E. and S. Livingston, London, 1962, 194. 3. Hartwell, J.L., Plants Used Against Cancer, Quarterman Publications, Laurence, MA, 1982, 108. 4. Wall, M.E. et al., Plant antitumor agents. 3. A convenient separation of tannins from other plant constituents, J. Pharm. Sci., 58, 839, 1969. 5. Palmer, E. and Pitman, N, Trees of Southern Africa, A.A. Balkema, Cape Town, 1972, Vol. 3. 6. Kokwaro, O., Medicinal Plants of East Africa, East African Literature Bureau, Nairobi, 1976. 7. Samuelsson, G. et al., Inventory of plants used in traditional medicine in Somalia. II. Plants of the families combretaceae to labiatae, J. Ethnopharm., 37, 47, 1992. 8. Iwu, M.M., Handbook of African Medicinal Plants, CRC Press, Boca Raton, FL, 1993. 9. Gurib-Fakim, A. and Brendler, T., Medicinal and Aromatic Plants of Indian Ocean Islands: Mada- gascar, Comoros, Seychelles, and Mascarenes, Medpharm Scientific Publishers, Stuttgart, Germany, 2004, 194, 463. 10. Duke, J., Medicinal Herbs, 2nd ed., CRC Press, New York, 2002, 539. 11. Pettit, G.R. et al., Antineoplastic agents 338. The cancer cell growth inhibitory. Constituents of terminalia arjuna (combretaceae), J. Ethnopharm., 53, 57, 1996. 12. Chinemana, F. et al., Indigenous plant remedies in Zimbabwe, J. Ethnopharm., 14, 159, 1985. 13. Cushman, M. et al., Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization, J. Med. Chem., 34, 2579, 1991. 14. Sackett, D.L., Podophyllotoxin, steganacin, and combretastatin: natural products that bind at the colchicine site of tubulin, Pharm. Ther., 59, 163, 1993. 15. Tozer, G.M. et al., The biology of the combretastatins as tumour vascular targeting agents. Int. J. Exp. Path., 83, 21, 2002. 16. Lin, C.M. et al., Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin: a structure-activity study, Mol. Pharm., 34, 200, 1988. 17. Li, Q. and Sham, H.L., Discovery and development of antimitotic agents that inhibit tubulin poly- merization for the treatment of cancer, Exp. Opin. Ther. Pat., 12, 1663, 2002. 18. Rowinsky, E.K. et al., Horizons in cancer therapeutics: from bench to bedside, Continuing Education, 3, 1, 2002. 19. Hamel, E., Antimitotic natural products and their interactions with tubulin, Med. Res. Rev., 16, 207, 1996. 20. Thorpe, P.E., Vascular targeting agents as cancer therapeutics, Clin. Cancer Res., 10, 415, 2004. Copyright © 2005 CRC Press, LLC 21. Griggs, J., Metcalfe, J.C., and Hesketh, R., Targeting tumour vasculature: the development of com- bretastatin A4, Lancet Onc., 2, 82, 2001. 22. Bibby, M.C., Combretastatin anticancer drugs, Drugs of the Future, 27, 475, 2002. 23. Cirla, A. and Mann, J., Combretastatins: from natural products to drug discovery, Nat. Prod. Rep., 20, 558, 2003. 24. Nam, N-H., Combretastatin A-4 analogues as antimitotic antitumor agents, Curr. Med. Chem., 10, 1697, 2003. 25. Pettit, G.R. and Lippert, J.W., Preparation of combretastatin A-1 phosphate and combretastatin B-1 phosphate prodrugs with increased solubility, PCT Int. Application WO2001081355 A1, 2001. 26. Pettit, G.R. and Minardi, M.D., Preparation of combretastatin A3 diphosphate prodrugs for the treatment of cancer, PCT Int. Application WO2002102766 A2, 2002. 27. Pettit, G.R. and Singh, S.B, Combretastatin A-4, US Patent 4,996,237, 1991. 28. Pettit, G.R. and Singh, S.B., Isolation, structural elucidation and synthesis of novel antineoplastic substances denominated “combretastatins,” US Patent 5,569,786, 1996. 29. Pettit, G. R., Combretastatin A-4 prodrug, US Patent 5,561,122, 1996. 30. Pinney, K. G. et al., Description of anti-mitotic agents which inhibit tubulin polymerization, US Patent 6,350,777, 2000. 31. Pettit, G.R. and Grealish, M.P., Synthesis of hydroxyphenstatin and the prodrugs thereof as anticancer and antimicrobial agents, PCT Int. Application WO2001081288 A1, 2001. 32. Pettit, G.R. and Brian, T., Synthesis and formulation of phenstatin and related prodrugs for use as antitumor agents, PCT Int. Application WO9934788 A1, 1999. 33. Pettit, G.R. and Rhodes, M.R., Preparation and formulation of combretastatin A4 prodrugs and their trans-isomers for use as antitumor agents, PCT Int. Application WO9935150 A1, 1999. 34. Pettit, G.R. and Moser, B.R., Preparation of combretastatin A-2 prodrugs as antitumor and antimi- crobial agents, PCT Int. Application WO2003059855 A1, 2003. 35. Kingston, D.G.I., Samaranayake, G., and Ivey, C.A., The chemistry of taxol, a clinically useful anticancer agent, J. Nat. Prod., 53, 1, 1990. 36. Nicolaou, K.C. et al., Synthesis of epothilones A and B in solid and solution phase, Nature, 387, 268, 1997. 37. Owellen, R.J. et al., Inhibition of tubulin-microtubule polymerization by drugs of the vinca alkaloid class, Cancer Res., 36, 1499, 1976. 38. Pettit, G.R. et al., The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10, J. Am. Chem. Soc., 109, 6883, 1987. 39. Pettit, G.R. et al., Antineoplastic agents 365. Dolastatin 10 SAR probes, Anticancer Drug Des., 13, 243, 1998. 40. Lin, C.M. et al., Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin, Biochemistry, 28, 6984, 1989. 41. Chaplin, D.J., Pettit, G.R., and Hill, S.A., Anti-vascular approaches to solid tumor therapy: evaluation of combretastatin A4 phosphate, Anticancer Res., 19, 189, 1999. 42. Jordon, A. et al., Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle, Med. Res. Rev., 18, 259, 1998. 43. Pinney, K.G. et al., A new anti-tubulin agent containing the benzo[b]thiophene ring system, Bioorg. Med. Chem. Lett., 9, 1081, 1999. 44. Pettit, G.R. and Lippert, J.W., Antineoplastic agents 429. Syntheses of the combretastatin A-1 and combretastatin B-1 prodrugs, Anticancer Drug Des., 15, 203, 2000. 45. Pettit, G.R. et al., Antineoplastic agents 322. Synthesis of combretastatin A-4 prodrugs, Anticancer Drug Des., 10, 299, 1995. 46. Pettit, G.R. et al., Isolation and structure of combretastatin, Can. J. Chem., 60, 1374, 1982. 47. Pettit, G.R., Cragg, G.M., and Singh, S.B., Antineoplastic agents, 122. Constituents of Combretum caffrum, J. Nat. Prod., 50, 386, 1987. 48. Pettit, G.R. et al., Isolation, structure, and synthesis of combretastatins A-1 and B-1, potent new inhibitors of microtubule assembly, derived from Combretum caffrum, J. Nat. Prod., 50, 119, 1987. 49. Pettit, G.R. and Singh, S.B., Isolation, structure, and synthesis of combretastatin A-2, A-3, and B-2, Can. J. Chem., 65, 2390, 1987. 50. Pettit, G.R. et al., Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-5, and A-6, J. Med. Chem., 38, 1666, 1995. Copyright © 2005 CRC Press, LLC 51. Pettit, G.R., Singh, S.B., and Schmidt, J.M., Isolation, structure, synthesis, and antimitotic properties of combretastatins B-3 and B-4 from Combretum caffrum, J. Nat. Prod., 51, 517, 1988. 52. Singh, S.B. and Pettit, G.R., Isolation, structure, and synthesis of combretastatin C-1, J. Org. Chem., 54, 4105, 1989. 53. Pettit, G.R., Singh, S.B., and Niven, M.L., Isolation and structure of combretastatin D-1: a cell growth inhibitory macrocyclic lactone from Combretum caffrum, J. Am. Chem. Soc., 110, 8539, 1988. 54. Singh, S.B. and Pettit, G.R., Antineoplastic agents. 206. Structure of the cytostatic macrocyclic lactone combretastatin D-2, J. Org. Chem., 55, 2797, 1990. 55. White, A., Principles of Biochemistry, 59th ed., McGraw-Hill, New York, 1978. 56. Nogales, E., Wolf, S.G., and Downing, K.H., Structure of the α,β-tubulin dimer by electron crystal- lography, Nature, 391, 199, 1998. 57. Rao, S., Horwitz, S.B., and Ringel, I., Direct photoaffinity labeling of tubulin with taxol, J. Natl. Cancer Inst., 84, 785, 1992. 58. Staretz, M.E. and Hastie, S.B., Synthesis, photochemical reactions, and tubulin binding of novel photoaffinity labeling derivatives of colchicine, J. Org. Chem., 58, 1589, 1993. 59. Hahn, K.M., Hastie, S.B., and Sundberg, R.J., Synthesis and evaluation of 2-diazo-3,3,3-trifluopro- panoyl derivatives of colchicine and podophyllotoxin as photoaffinity labels: reactivity, photochem- istry, and tubulin binding, Photochem. Photobiol., 55, 17, 1992. 60. Wolff, J. et al., Direct photoaffinity labeling of tubulin with colchicine, Proc. Natl. Acad. Sci. USA, 88, 2820, 1991. 61. Floyd, L.J., Barnes, L.D., and Williams, R.F., Photoaffinity labeling of tubulin with (2-nitro-4- azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites, Biochemistry, 28, 8515, 1989. 62. Bai, R. et al., Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine, J. Biol. Chem., 271, 12639, 1996. 63. Olszewski, J. D. et al., Potential photoaffinity labels for tubulin. Synthesis and

evaluation of diazo- cyclohexadienone and azide analogs of colchicine, combretastatin, and 3,4,5-trimethoxybiphenyl, J. Org. Chem., 59, 4285, 1994. 64. Pinney, K.G. et al., Synthesis and biological evaluation of aryl azide derivatives of combretastatin A- 4 as molecular probes for tubulin, Bioorg. Med. Chem., 8, 2417, 2000. 65. Sawada, T. et al., A fluorescent probe and a photoaffinity labeling reagent to study the binding site of maytansine and rhizoxin on tubulin, Bioconj. Chem., 4, 284, 1993. 66. Sawada, T. et al., Identification of the fragment photoaffinity-labeled with azidodansyl-rhizoxin as Met-363-Lys-379 on beta-tubulin, Biochem. Pharm., 45, 1387, 1993. 67. Sawada, T. et al., Fluorescent and photoaffinity labeling derivatives of rhizoxin, Biochem. Biophys. Res. Comm., 178, 558, 1991. 68. Chavan, A.J. et al., Forskolin photoaffinity probes for the evaluation of tubulin binding sites, Bioconj. Chem., 4, 268, 1993. 69. Safa, A.R., Hamel, E., and Felsted, R.L., Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine, Biochemistry, 26, 97, 1987. 70. Williams, R.F. et al., A photoaffinity derivative of colchicine: 6-(4-azido-2-nitrophenylamino)hex- anoyldeacetylcolchicine; photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin, J. Biol. Chem., 260, 13794, 1985. 71. Nogales, E., Structural insights into microtubule function, Ann. Rev. Biochem., 69, 277, 2000. 72. Hadimani, M. et al., 2-(3-tert-Butyldimethylsiloxy-4-methoxyphenyl)-6-methoxy-3-(3,4,5-trimethox- ybenzoyl)indole, Acta Cryst., Sec. C Crystal Struct. Comm., C58, 330, 2002. 73. Siemann, D.W., Vascular targeting agents: an introduction, Int. J. Radiat. Oncol. Biol. Phys., 54, 1472, 2002. 74. Chaplin, D.J. and Hill, S.A., The development of combretastatin A4 phosphate as a vascular targeting agent, Int. J. Radiat. Oncol. Biol. Phys., 54, 1491, 2002. 75. Thorpe, P.E., Chaplin, D.J., and Blakeley, D.C., The first international conference on vascular targeting: meeting overview, Cancer Res., 63, 1144, 2003. 76. Chaplin, D.J. and Dougherty, G.J., Tumour vasculature as a target for cancer therapy, Br. J. Cancer, 80, 57, 1999. Copyright © 2005 CRC Press, LLC 77. Kanthou, C. and Tozer, G.M., The tumor vascular targeting agent combretastatin A-4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells, Blood, 99, 2060, 2002. 78. Tozer, G.M. et al., Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability, Cancer Res., 61, 6413, 2001. 79. Galbraith, S.M. et al., Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumor vascular targeting activity in vivo, Anticancer Res., 21, 93, 2001. 80. Young, S. and Chaplin, D.J., Combretastin A4 phosphate: background and current clinical status, Expert Opin. Investig. Drugs, 13, 1171, 2004. 81. Pettit, G.R. et al., Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4, Experientia, 45, 209, 1989. 82. Pettit, G.R. and Rhodes, M.R., Antineoplastic agents 389. New syntheses of the combretastatin A-4 prodrug, Anticancer Drug Des., 13, 183, 1998. 83. Boger, D.L., Sakya, S.M., and Yohannes, D., Total synthesis of combretastatin D-2: intramolecular Ullmann macrocyclization reaction, J. Org. Chem., 56, 4204, 1991. 84. Deleted from text. 85. Gaukroger, K. et al., Novel syntheses of cis and trans isomers of combretastatin A-4, J. Org. Chem., 66, 8135, 2001. 86. Bui, V.P. et al., Direct biooxidation of arenes to corresponding catechols with E. coli JM109 (PDTG602). Application to synthesis of combretastatins A-1 and B-1, Tetrahedron Lett., 43, 2839, 2002. 87. Lawrence, N.J. et al., The synthesis of (E) and (Z)-combretastatins A-4 and a phenanthrene from Combretum caffrum, Synthesis, 9, 1656, 1999. 88. Deshpande, V.H. and Gokhale, N.J., Synthesis of combretastatin D-2, Tetrahedron Lett., 33, 4213, 1992. 89. Couladouros, E.A. and Soufli, I.C., Total synthesis of natural (-)-combretastatin D-1, Tetrahedron Lett., 36, 9369, 1995. 90. Couladouros, E.A. and Soufli, I.C., Synthesis of combretastatin D-2. An efficient route to caffrane macrolactones, Tetrahedron Lett., 35, 4409, 1994. 91. Chaplin, D.J. et al., Functionalized stilbene derivatives as improved vascular targeting agents, US Patent 20030149003 A1, 2003. 92. Ohsumi, K. et al., Novel combretastatin analogues effective against murine solid tumors: design and structure-activity relationships, J. Med. Chem., 41, 3022, 1998. 93. Ohsumi, K. et al., Synthesis and antitumor activities of amino acid prodrugs of amino-combretastatins, Anticancer Drug Des., 14, 539, 1999. 94. Siles, R., Hadimani, M., and Pinney, K.G., Cancer chemotherapy: prodrug strategies for enhanced bioavailability and selectivity of vascular targeting agents, 58th Southwest Regional Meeting of The American Chemical Society (abstract 236), Austin, TX, November 4, 2002. 95. Pettit, G.R. et al., Antineoplastic agents. 487. Synthesis and biological evaluation of the antineoplastic agent 3,4-methylenedioxy-5,4-dimethoxy-3-amino-Z-stilbene and derived amino acid amides, J. Med. Chem., 46, 525, 2003. 96. Lawrence, N.J. et al., Synthesis and anticancer activity of fluorinated analogues of combretastatin A- 4, J. Fluorine Chem., 123, 101, 2003. 97. Davis, P.D., Compositions with vascular damaging activity, PCT Int. Application WO0214329 A1, 2002. 98. Gaukroger, K., Structural requirements for the interaction of combretastatins with tubulin: how important is the trimethoxy unit? Org. Biomol. Chem., 1, 3033, 2003. 99. Pettit, G.R. et al., Antineoplastic agents. 379. Synthesis of phenstatin phosphate, J. Med. Chem., 41, 1688, 1998. 100. Pettit, G.R. et al., Antineoplastic agents. 443. Synthesis of the cancer cell growth inhibitor hydrox- yphenstatin and its sodium diphosphate prodrug, J. Med. Chem., 43, 2731, 2000. 101. Medarde, M. et al., Synthesis and antineoplastic activity of combretastatin analogues: heterocombre- tastatins, European J. Med. Chem., 33, 71, 1998. 102. Andres, C.J. et al., “Combretatropones” — hybrids of combretastatin and colchicine. Synthesis and biochemical evaluation, Bioorg. Med. Chem. Lett., 3, 565, 1993. Copyright © 2005 CRC Press, LLC 103. Janik, M.E. and Bane, S.L., Synthesis and antimicrotubule activity of combretatropone derivatives, Bioorg. Med. Chem., 10, 1895, 2002. 104. Shirai, R. et al., Synthesis and anti-tubulin activity of aza-combretastatins, Bioorg. Med. Chem. Lett., 4, 699, 1994. 105. Pettit, G.R. et al., Antineoplastic agents. 410. Asymmetric hydroxylation of trans-combretastatin A- 4, J. Med. Chem., 42, 1459, 1999. 106. Pettit, G.R. et al., Antineoplastic agents 440. Asymmetric synthesis and evaluation of the combret- astatin A-1 SAR probes (1S, 2S)-and (1R, 2R)-1,2-dihydroxy-1-(2,3-dihydroxy-4-methoxyphenyl)-2- (3,4,5-trimethoxyphenyl)-ethane, J. Nat. Prod., 63, 969, 2000. 107. Shirai, R. et al., Asymmetric synthesis of antimitotic combretadioxolane with potent antitumor activity against multi-drug resistant cells, Bioorg. Med. Chem. Lett., 8, 1997, 1998. 108. Pettit, G.R. et al., Antineoplastic agents 442. Synthesis and biological activities of dioxostatin, Anti- cancer Drug Des., 15, 361, 2000. 109. Shirai, R., Okabe, T., and Iwasaki, S., Synthesis of comformationally restricted combretastatins, Heterocycles, 46, 145, 1997. 110. Ducki, S. et al., Potent antimitotic and cell growth inhibitory properties of substituted chalcones, Bioorg. Med. Chem. Lett., 8, 1051, 1998. 111. Wang, L. et al., Potent, orally active heterocycle-based combretastatin A-4 analogues: synthesis, structure-activity relationship, pharmacokinetics, and in vivo antitumor activity evaluation, J. Med. Chem., 45, 1697, 2002. 112. Hatanaka, T. et al., Novel B ring modified combretastatin analogues: syntheses and antineoplastic activity, Bioorg. Med. Chem. Lett., 8, 3371, 1998. 113. Medina, J.C. et al., Pentafluorobenzenesulfonamides and analogs, US Patent 5880151, 1999. 114. Flygare, J.A. et al., Pentafluorobenzenesulfonamides and analogs, US Patent 6121304, 2000. 115. Pinney, K.G., Wang, F., and Del Pilar Mejia, M., Preparation of indole-containing and combretastatin- related anti-mitotic and anti-tubulin polymerization agents, PCT Int. Application WO2001019794 A2, 2001. 116. Flynn, B.L., Hamel, E., and Jung, M.K., One-pot synthesis of benzo[b]furan and indole inhibitors of tubulin polymerization, J. Med. Chem., 45, 2670, 2002. 117. Gastpar, R. et al., Methoxy-substituted 3-formyl-2-phenylindoles inhibit tubulin polymerization, J. Med. Chem., 41, 4965, 1998. 118. Chen, Z. et al., Preparation of new anti-tubulin ligands through a dual-mode, addition-elimination reaction to a bromo-substituted,-unsaturated sulfoxide, J. Org. Chem., 65, 8811, 2000. 119. Pinney, K.G. et al., Preparation of trimethoxyphenyl-containing tubulin binding ligands and corre- sponding prodrug constructs as inhibitors of tubulin polymerization and antimitotic agents, PCT Int. Application WO2001068654, 2001. 120. Pinney, K.G. et al., Anti-mitotic agents which inhibit tubulin polymerization, US Patent 6162930, 2000. 121. Pinney, K.G. et al., Anti-mitotic agents which inhibit tubulin polymerization, Australian Patent 732917, 2001. 122. Flynn, B.L. et al., The synthesis and tubulin binding activity of thiophene-based analogues of com- bretastatin A-4, Bioorg. Med. Chem. Lett., 11, 2341, 2001. 123. Flynn, B.L., Verdier-Pinard, P., and Hamel, E., A novel palladium-mediated coupling approach to 2,3- disubstituted benzo[b]thiophenes and its application to the synthesis of tubulin binding agents, Org. Lett., 3, 651, 2001. 124. Pinney, K.G. et al., Preparation of aryl and arylcarbonylbenzothiophenes, -benzofurans, -indenes, and indoles as tubulin binding ligands and corresponding prodrug constructs thereof useful as antitumor agents, US Patent 2004044059, 2004. 125. Flynn, B.L. and Hamel, E., Synthesis of indoles, benzofurans, and related compounds as potential tubulin binding agents, PCT Int. Application WO2002060872 A1, 2002. 126. Pinney, K.G. et al., Tubulin binding ligands and corresponding prodrug constructs, US Patent 6593374, 2003. 127. Ghatak, A. et al., Synthesis of methoxy and hydroxy containing tetralones: versatile intermediates for the preparation of biologically relevant molecules, Tetrahedron Lett., 44, 4145, 2003. 128. Goldbrunner, M. et al., Inhibition of tubulin polymerization by 5,6-dihydroindolo[2,1-α]isoquinoline derivatives, J. Med. Chem., 40, 3524, 1997. Copyright © 2005 CRC Press, LLC 129. Li, Q. et al., Synthesis and biological evaluation of 2-indolyloxazolines as a new class of tubulin polymerization inhibitors. Discovery of A-289099 as an orally active antitumor agent, Bioorg. Med. Chem. Lett., 12, 465, 2002. 130. Tahir, S.K. et al., Biological activity of A-289099: an orally active tubulin-binding indolyloxazoline derivative, Mol. Cancer Ther., 2, 227, 2003. 131. Hori, K. and Saito, S., Microvascular mechanisms by which the combretastatin A-4 derivative AC7700 (AVE8062) induces tumour blood flow stasis, Br. J. Cancer, 89, 1334, 2003. 132. Blakey, D.C. et al., ZD6126: a novel small molecule vascular targeting agent, Int. J. Radiat. Onc. Biol. Phys., 54, 1497, 2002. 133. Siemann, D.W. and Rojiani, A.M., Antitumor efficacy of conventional anticancer drugs is enhanced by the vascular targeting agent ZD6126, Int. J. Radiat. Oncol. Biol. Phys., 54, 1512, 2002. 134. Siemann, D.W. and Rojiani, A.M., Enhancement of radiation therapy by the novel vascular targeting agent ZD6126, Int. J. Radiat. Oncol. Biol. Phys., 53, 164, 2002. 135. Micheletti, G. et al., Vascular-targeting activity of ZD6126, a novel tubulin-binding agent, Cancer Res., 63, 1534, 2003. 136. Chaplin, D.J. et al., Antivascular approaches to solid tumour therapy: evaluation of tubulin binding agents, Br. J. Cancer, 27, S86, 1996. 137. Dark, G.D. et al., Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature, Cancer Res., 57, 1829, 1997. 138. West, C.M.L. and Price, P., Combretastatin A4 phosphate, Anticancer Drugs, 15, 179, 2004. 139. McGown, A.T. and Fox, B.W., Structural and biochemical comparison of the anti-mitotic agents colchicine, combretastatin A4 and amphethinile, Anticancer Drug Des., 3, 249, 1989. 140. Hamel, E. and Lin, C.M., Interactions of combretastatin, a new plant-derived antimitotic agent, with tubulin, Biochem. Pharm., 32, 3864, 1983. 141. Horsman, M.R. et al., The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors, Int. J. Radiat. Oncol. Biol. Phys., 42, 895, 1998. 142. Li, L., Rojiani, A., and Siemann, D.W., Targeting the tumor vasculature with combretastatin A-4 disodium phosphate: effects on radiation therapy, Int. J. Radiat. Oncol. Biol. Phys., 42, 899, 1998. 143. Tozer, G.M., et al., Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues, Cancer Res., 57, 1829, 1997. 144. Grosios, K. et al., In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug, Br. J. Cancer, 81, 1318, 1999. 145. Beauregard, D.A. et al., Magnetic resonance imaging and spectroscopy of combretastatin A4 prodrug- induced disruption of tumour perfusion and energetic status, Br. J. Cancer, 77, 1761, 1998. 146. Eikesdal, H.P. et al., The new tubulin-inhibitor combretastatin A-4 enhances thermal damage in the BT4An rat glioma, Int. J. Radiat. Oncol. Biol. Phys., 46, 645, 2000. 147. Maxwell, R.J. et al., Effects of combretastatin on murine tumours monitored by 31P MRS, 1H MRS and 1H MRI, Int. J. Radiat. Oncol. Biol. Phys., 42, 891, 1998. 148. Ahmed, B. et al., Vascular targeting effect of combretastatin A-4 phosphate dominates the inherent angiogenesis inhibitory activity, Int. J. Cancer, 105, 20, 2003. 149. Chaplin, D.J. and Horsman, M.R., The influence of tumour temperature on ischemia-induced cell death: potential implications for the evaluation of vascular mediated therapies, Radiother. Oncol., 30, 59, 1994. 150. Siemann, D.W. et al., Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy, Int. J. Cancer, 99, 1, 2002. 151. Chaplin, D.J. et al., Therapeutic significance of microenvironmental factors, Medical Radiology, (Radiation Oncology): Blood Perfusion and

Microenvironment of Human Tumors, Springer, Berlin, 1998, 133. 152. Horsman, M.R. et al., Combretastatins: novel vascular targeting drugs for improving anti- cancer therapy. Combretastatins and conventional therapy, Adv. Exp. Med. Biol., 476, 311, 2000. 153. Landuyt, W. et al., In vivo antitumor effect of vascular targeting combined with either ionizing radiation or anti-angiogenesis treatment, Int. J. Radiat. Oncol. Biol. Phys., 49, 443, 2001. 154. Horsman, M.R. and Murata, R., Combination of vascular targeting agents with thermal or radiation therapy, Int. J. Radiation Oncol. Biol. Phys., 54, 1518, 2002. Copyright © 2005 CRC Press, LLC 155. Pedley, R.B. et al., Eradication of colorectal xenografts by combined radioimmunotherapy and com- bretastatin A-4 3-O-phosphate, Cancer Res., 61, 4716, 2001. 156. Grosios, K. et al., Combination chemotherapy with combretastatin A-4 phosphate and 5-fluorouracil in an experimental murine colon adenocarcinoma, Anticancer Res., 20, 229, 2000. 157. Nelkin, B.D. and Ball, D.W., Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma, Oncol. Rep., 8, 157, 2001. 158. Wildiers, H. et al., Combretastatin A-4 phosphate enhances CPT-11 activity independently of the administration sequence, Eur. J. Cancer, 40, 284, 2004. 159. Murata, R. et al., Interaction between combretastatin A-4 disodium phosphate and radiation in murine tumors, Radiother. Oncol., 60, 155, 2001. 160. Murata, R., Overgaard, J., and Horsman, M.R., Combretastatin A-4 disodium phosphate: a vascular targeting agent that improves that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy, Int. J. Radiat. Oncol. Biol. Phys., 51, 1018, 2001. 161. Horsman, M.R., Christensen, K.L., and Overgaard, J., Hydralazine-induced enhancement of hyper- thermic damage in a C3H mammary carcinoma in vivo, Int. J. Hyperthermia, 5, 123, 1989. 162. Eikesdal, H.P. et al., Combretastatin A-4 and hyperthermia; a potent combination for the treatment of solid tumors, Radiother. Oncol., 60, 147, 2001. 163. Holwell, S.E. et al., Anti-tumor and anti-vascular effects of the novel tubulin-binding agent combre- tastatin A-1 phosphate, Anticancer Res., 22, 3933, 2002. 164. Hill, S.A. et al., Preclinical evaluation of the antitumour activity of the novel vascular targeting agent OXi 4503, Anticancer Res., 22, 1453, 2002. 165. Shnyder, S.D. et al., Combretastatin A-1 phosphate potentiates the antitumour activity of cisplatin in a murine adenocarcinoma model, Anticancer Res., 23, 1619, 2003. 166. Hua, J. et al., Oxi4503, a novel vascular targeting agent: effects on blood flow and antitumor activity in comparison to combretastatin A-4 phosphate, Anticancer Res., 23, 1433, 2003. 167. Kirwan, I.G. et al., Comparative preclinical pharmacokinetic and metabolic studies of the combret- astatin prodrugs combretastatin A4 phosphate and A1 phosphate, Clin. Cancer Res., 10, 1446, 2004. 168. Dowlati, A. et al., A phase 1b pharmacokinetic and translational study of the novel vascular targeting agent combretastatin A-4 phosphate on a single-dose intravenous schedule in patients with advanced cancer, Cancer Res., 62, 3408, 2002. 169. Stevenson, J.P. et al., Phase I trial of the antivascular agent combretastatin A4 phosphate on a 5-day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow, J. Clin. Oncol., 21, 4428, 2003. 170. Rustin, G.J. et al., Phase I clinical trial of weekly combretastatin A4 phosphate: clinical and pharma- cokinetic results, J. Clin. Oncol., 21, 2815, 2003. 171. Galbraith, S.M. et al., Combretastatin A4 phosphate has tumor antivascular activity in rat and man as demonstrated by dynamic magnetic resonance imaging, J. Clin. Oncol., 21, 2831, 2003. 172. Anderson, H.L. et al., Assessment of pharmacodynamic vascular response in a phase I trial of combretastatin A4 phosphate, J. Clin. Oncol., 21, 2823, 2003. Copyright © 2005 CRC Press, LLC 4 Homoharringtonine and Related Compounds Hideji Itokawa, Xihong Wang, and Kuo-Hsiung Lee CONTENTS I. Introduction II. Structures of the Drug and Related Compounds A. Isolation of New Cephalotaxus Alkaloids B. Modification of the Skeleton of Homoharringtonine (HHT) through Unusual Rearrangements III. Synthesis of the Drug A. Synthesis of Acyl Groups B. Total Synthesis IV. Mechanism of Action V. Medicinal Chemistry of the Drug VI. Development of the Clinical Drug from the Natural Products Lead VII. Clinical Applications A. Chinese Clinical Trials Treating Leukemias with Cephalotaxine Esters B. Phase I Studies in the United States C. Phase II Studies in Solid Tumors D. Phase I–II Studies in Patients with Acute Leukemia E. Phase II Studies in Myelodysplastic Syndrome (MDS) F. Studies in Acute Promyelocytic Leukemia (APL) G. Phase II Studies in Chronic Myeloid Leukemia (CML) Patients VIII. Conclusions Acknowledgments References I. INTRODUCTION The genus Cephalotaxus contains coniferous evergreen trees and shrubs that are indigenous to Asia. Historically, the bark has long been used in China as traditional medicine for a variety of indications. Through screening assays and preliminary clinical trials, Chinese investigators discovered that the total alkaloid fraction from Cephalotaxus fortunei Hook. F possessed definite antitumor activity.1 Homoharringtonine (1, HHT) (cephalotaxine 4-methyl-2-hydroxy-4-methylpentyl butanedioate), one of the most active Cephalotaxus alkaloids, was obtained by alcoholic extraction from the Chinese evergreen tree Cephalotaxus harringtonia K. Koch var. harringtonia.2 Other Cephalotaxus alkaloids have been isolated from various Cephalotaxus spp.3–5 The parent compound cephalotaxine (2) was first isolated by Paudler et al.3 from two Cephalotaxus species. Their initial structural proposal was later revised, and Abraham et al.6 proposed the correct structure and relative stereochemistry of 2 through x-ray crystallographic analysis of cephalotaxine Copyright © 2005 CRC Press, LLC O O O O O 1, R = 2, R = H 3, R = 4, R = H 5, R = N OH O MeO MeO MeO MeO H HO H OHO HO OH OH OH RO O O OMe O STRUCTURES 1–5 methiodide. The parent compound and its esters represent a group of alkaloids with a unique ring system.7–12 Although cephalotaxine is devoid of antitumor activity, ester alkaloids derived from ceph- alotaxine are of particular anticancer interest. The most active compounds in this series are HHT (1) and harringtonine (3),8 isolated by Powell et al.9 In addition to 1 and 3, the active ester alkaloids isoharringtonine (4) and deoxyharringtonine (5) have also been isolated from C. harringtonia.8 HHT (1) has been investigated in phase II clinical trials as an anticancer agent at the National Cancer Institute, Bethesda, Maryland. However, its side effects still remain an issue. Work has thus continued on the study of new components from this plant species and on the development of new analogs on the basis of the structure–activity relationships of these alkaloids. This chapter gives an overview of this work. The HHT alkaloids and related cephalotaxine esters have been reviewed previously by Cassady and Douros13 and by Kantarijian et al.,14 who selected HHT as the best compound to develop as an anticancer agent. II. STRUCTURES OF THE DRUG AND RELATED COMPOUNDS A. ISOLATION OF NEW CEPHALOTAXUS ALKALOIDS In the last several years, more than 20 Cephalotaxus alkaloids have been isolated from various Cephalotaxus spp.15–20 Cephalotaxus alkaloids isolated by Takano et al.15–20 were classified into three types based on the side chain structure. One group contains compounds having a carboxyl group at the end of side chain (e.g., 5′-des-O-methylharringtonine [6], 5′-des-O-methylhomoharringtonine [7], 5′-des-O-methylisoharringtonine [8] and 3′S-hydroxy-5′-des-O-methylharringtonine [9]). A second group contains compounds that vary by the number of methylene groups in the side chain (e.g., nordeoxyharringtonine [10], homodeoxyharringtonine [11], and bishomodeoxyharringtonine [12]). The third group of compounds contains terminal aromatic rings in the side chain (e.g., neoharringto- nine [13], homoneoharringtonine [14] and 3S-hydroxyneoharringtonine [15]).15–20 Cephalezomines C-F (16–19) were isolated by Morita et al.,21 and four new Cephalotaxus alkaloids, cephalotaxine α–N-oxide (20), cephalotaxine β-N-oxide (21), 11-β-hydroxycephalotax- ine β-N-oxide (22), and isocephalotaxine (23), together with several known alkaloids, were isolated recently from an EtOAc extract of the fruits of C. fortunei.22 These latter four compounds displayed O O O O H H 7, R = 6, R = HO HO 8, R = HO OH OH 9, R = HO HO HO OH O OH O OH HO OH O O O O O O O 10 R = 13, R = N MeO 11, R = 12, R = MeO MeO MeO O H C6H5 OH OH H O OH O O OHO OROMe O O 14, R = 15 H MeO , R = OH MeO C C 6H5 6H5 OH O OH O STRUCTURES 6–15 Copyright © 2005 CRC Press, LLC O O H O O O OH H H H N 16, R = 17, R = OH 18, R = OH 19, R = OH MeO MeO MeO MeO O HO H H HO OHO HO OH OH OH O O O OR OMe HO 17 O 10 O- O O- O O- O O N+ 8 N+ N N N+ O 5 7 O O O O 14 H H H 4 6 1 H 3 2 H H HO HO MeO HO OMe19 OMe HO OMe OH O 20 21 22 23 24 STRUCTURES 16–24 weak cytotoxicity against KB cells with IC50 values of 30, 14, 31, and 15 µg/mL, respectively.22 Desmethylcephalotaxinone (24) was obtained from C. harringtonia species.12 Other related compounds have a hydroxyl group at carbon-11 (e.g., 11α-hydroxyhomodeoxyhar- ringtonine [25], 11β-hydroxyhomodeoxyharringtonine [26], and 11β-hydroxydeoxyharringtonine [27]).19 In addition, the ester alkaloid drupangtonine (28) has a drupacine (32) skeleton.15 Two additional alkaloids, cephalezomines A (29) and B (30), were obtained from C. harringtonia f. drupacea.21 The Japanese species C. harringtonia (Knight) Koch. f. drupacea (Sieb. & Zucc.) Kitamura [= C. harringtonia var. drupacea (Sieb. & Zucc.) = C. drupacea Sieb. & Zucc.] was reported to contain the characteristic Cephalotaxus alkaloids HHT (1), cephalotaxine (2), harringtonine (3), isoharringtonine (4), deoxyharringtonine (5), and drupacine (32). 11-Hydroxycephalotaxine (31) and drupacine (32) were isolated from C. harringtonia var. drupacea,11 and compound 31 was converted to 32 under acidic conditions. The dimeric cephalotaxidine (33), obtained by Takano et al.,18 and cephalocyclidin A (34),23 are examples of unusual Cephalotaxus alkaloids. Epischellham- mericine,24 and 3-epischellhammericine25 were also obtained, but they belong to the homoerythrina alkaloid class.26 R2 O O O O N MeO MeO O 25 R1 = 26 R1 = H MeO 27 R1 = 31 R1 = H, R2 = β-OH H OH OH OH R1O O O O OMe R2 = α-OH R2 = β-OH R2 = β-OH H O O 28 R = 29 R = O O MeO 30 R = 32 R = H O N MeO HO MeO O H OH H OH O O OH RO O OMe O OH O MeO O O MeOH OMe O HO OH H OH OO OH H N H O HO O O O HO H N O MeO O 33 34 STRUCTURES 25–34 Copyright © 2005 CRC Press, LLC Arora et al.27,28 determined the absolute stereochemistry of cephalotaxine through x-ray crys- tallography. Working with the p-bromo-benzoate of 2, the researchers ascertained that natural (-)- cephalotaxine has an 3S,4S,5R configuration. B. MODIFICATION OF THE SKELETON OF HOMOHARRINGTONINE (HHT) THROUGH UNUSUAL REARRANGEMENTS29 Early biological evaluation indicated that the acyl group of 1 is very important for the activity of Cephalotaxus-type alkaloids. Numerous minor alkaloids possessing different ester groups have been isolated from Cephalotaxus plants, and previous efforts toward the modification of Cephalo- taxus alkaloids addressed the replacement of the parent ester moiety of 1 by various acyl groups. Several skeleton-modified analogs of HHT (1), which is the most abundant ester-type Cepha- lotaxus alkaloid showing potent antitumor activity, have been designed and synthesized. In addition, because the nitrogen lone pair might play an important role in expressing the activity, this region was selected for modification. Oxidation of 1 with hydrogen peroxide in methanol gave β-N-oxide 35 and α-N-oxide 36 in 26% and 36% yield, respectively (Figure 4.1). When a 1,2-dimethoxyethane solution of β-N-oxide 35 was heated in a sealed tube at 105˚C for 2 h, compound 37 and two unexpected compounds, 38 and 39, were obtained in yields of 37%, 44%, and 7.7%, respectively. Heating α-N-oxide 36 under the same conditions also gave compounds 37 (32%), 38 (36%), and 39 (7.6%). Zinc and acetic acid reduction of 38 and 39 gave ring contracted HHT analogs 40 and 41 in yields of 96% and 67%, respectively. Their stereostructures were confirmed from NOESY spectral data. III. SYNTHESIS OF THE DRUG A. SYNTHESIS OF ACYL GROUPS Alkaline hydrolysis of 1 or 3–5 yields cephalotaxine 2 plus a carboxylic acid moiety. Initially, the structures of the resulting dicarboxylic acids were identified by nuclear magnetic resonance and mass spectral studies.9,10 The structure of 42, the dimethyl ester

of the acid moiety from 5, was also verified by synthesis. Thus, methyl isopentyl ketone (43) was condensed with diethyl carbonate (44) to give ethyl 6-methyl-3-oxo-heptanoate (45), which was converted to a cyanohydrin (46). Acid catalyzed methanolysis of 46 provided a racemic dimethyl ester (42) identical with the product from 5, except for its chiral properties (Figure 4.2).10 This confirmatory synthesis supported structures assigned to the corresponding acid moieties of 1, 3, and 4. However, characterization of these dicarboxylic acids did not establish which of their two available carboxyl groups was attached to the cephalotaxine moiety. This feature was revealed through an attempted synthesis of 5. When cephalotaxine was acylated with the requisite half ester of 42, which had the primary carboxyl group in the acid chloride form, the resulting product was an isomer of 5, ψ-deoxyharringtonine (47). The nuclear magnetic resonance spectrum of 47 was distinctly different from that of deoxyharringtonine (5), thus providing evidence for structure 5, in which the ester side chain is attached through the tertiary carboxyl group. From close parallels of the nuclear magnetic resonance spectra, Mikolajczak and associates10 concluded that the acyl moiety was constituted similarly in 1 and 3–5. This conclusion was reinforced by additional synthesis of the dicarboxylic acid moieties. Auerbach and coworkers30 carried out an alternative synthesis of 42 (Figure 4.3). Methyl itaconate (48) was epoxidized with buffered peroxytrifluoroacetic acid, and the resulting epoxide (49) was condensed with a reagent prepared from isobutyl lithium and cuprous iodide. The resulting product was 42. Ipaktchi and Weinreb31 synthesized two diastereomeric compounds corresponding to the acid moiety of isoharringtonine (4). Isoamylacetoacetate (50) was converted through a haloform-type reaction to isoamylfumaric acid (51), which, in turn, was dehydrated to an anhydride. This anhydride Copyright © 2005 CRC Press, LLC 1 H2O2/MeOH H b H b H H b b O O- O O- b H b a N N ⊕ H a ⊕ O O H H H H RO RO 35 OMe 36 OMe a b Cope Reaction H O- O O O N :N O O H H ⊕ H H H RO RO OMe OMe Reverse Cope Reaction O 16 - N Me O O ⊕ N O 1 O H 20 H 4 2 H O H H H RO H 37 NOE NOE RO OMe Zn, AcOH Me H NOE H Me O NOE O N O N N 15 O O O O 1 H H 19 O H 2 H H H H H 1 RO OMe 38 RO 39 OMe RO OMe Zn, AcOH Zn, AcOH O Me H H Me R = MeO 9 O HO O 8 N N 6 O O O H OH 13 H 1 3 H H 40 RO OMe 41 RO OMe FIGURE 4.1 Modification of the skeleton of HHT through unusual rearrangements. was converted to a dimethyl maleate derivative, which was reacted with osmium tetroxide-hydrogen peroxide in t-butanol to give a diol (52). This diol was identical with the dimethyl ester from 4, except for optical rotation. Thus, the relative configuration of 52 was established as erythro, although the absolute configuration had yet to be determined (Figure 4.4). Kelly and coworkers32 synthesized the diacid from hydrolysis of harringtonine (3) through an α-keto ester intermediate (54) (Figure 4.4). Benzylated hydroxyacetylene 53 was treated with Copyright © 2005 CRC Press, LLC O O CH + 2 EtO OEt 43 H 44 NaH O O OEt 45 HCN OH O OEt CN OH O 46 OR COOR 42 R = Me 42a R = H FIGURE 4.2 Synthesis of deoxyharringtonine side chain. O N O H H OH O OMe O O OMe 47 O O O O OH MeO MeO MeO OMe OMe OMe O 48 O 49 42 O FIGURE 4.3 Auerbach synthesis of deoxyharringtonine side chain. OH COOEt COOEt COOEt O H CH3 HOOC H COOMe OH 50 51 52 O C6H5CH2O H C6H5CH2O O 53 54 O OH OH COOMe OHO C6H5CH2O O 56 COOMe 55 COOMe O O O Br + COOMe OMe 57 58 59 OH OH COOMe COOMe OH CN 61 COOH 60 FIGURE 4.4 Additional synthesis of side chain acids. Copyright © 2005 CRC Press, LLC butyllithium, and the resulting lithio derivative was condensed with ethyl t-butyl oxalate to provide the α-keto ester 54. At this stage, the carbomethoxymethyl side chain was introduced by condensing 54 with lithium methyl acetate (LiCH2CO2Me). Debutylation of the product 55 was effected with trifluoroacetic acid. After methylation with diazomethane and debenzylation by hydrogenolysis, the dimethyl ester (56) of the desired diacid was obtained. Utawanit33 synthesized the diacid resulting from hydrolysis of HHT (1). 1-Bromo-3-methyl-2- butene (57) was condensed with methyl acetoacetate (58) to give (59) (Figure 4.4). This unsaturated ester (59) was converted to the corresponding cyanohydrin (60), and subsequent acid hydrolysis gave the racemic form of the desired diacid (61). Hydration of the double bond was accompanied by hydrolysis of the nitrite group. B. TOTAL SYNTHESIS The unique structure and the therapeutic potential of this group of alkaloids have stimulated many syntheses of cephalotaxine (CET, 2),34–37 as well as numerous studies on the construction of the pentacyclic ring system.38–43 The unique heterocyclic ABCDE ring system of CET has continued to be a proving ground for new synthetic strategies and methods. Excellent review articles on this subject have been published by Huang and Xue,44 Jalil Miah et al.,34 and most recently, by Li and Wang.45 Total synthesis of CET was accomplished on the basis of a conceptually novel strategy that featured key transannular reductive skeletal rearrangements to construct the pentacyclic ring skeleton. The synthetic potential of the designated Clemments–Clemo–Prelog–Leonard reductive rearrange- ment was demonstrated for the first time in a facile synthesis of the benzazepine subunit of CET. A novel endocyclic enamine (cyclopentenone) annulation was discovered as an unusual azo-Nazarov- type cyclization. A key pentacyclic precursor, ketone 62, was based on a transannular skeletal rearrangement (Figure 4.5, path a) of the cyclic enone 63.46 This pathway parallels the biogenesis of CET that was postulated by Parry et al. (Figure 4.6).47 Enone 63 could in turn be derived from cyclic ketone 65. A more conventional strategy is outlined in Figure 4.5, path b, which involves a transannular rearrangement of ketone 65 to benzazepine precursor 64. The synthesis of 62 is shown in Figure 4.7.46 β-(3, 4-Methylenedioxy)-phenethylamine (66) was converted to 67 in 62% overall yield by the following sequence: (1) Bischler–Napiralsky cyclization of corresponding oxalamide, (2) catalytic hydrogenation, and (3) N-alkylation with ethyl 4-bromobutyrate. Reaction of diester 67 with allyl bromide in DMSO gave the allylation product O O O H N N a O O N O HO OCH 62 O 63 3 O 2 b O O N N O O O HO 64 65 FIGURE 4.5 Retrosynthetic strategic plan for synthesis of 2. Copyright © 2005 CRC Press, LLC HO HO a HN HO a N b HO HO HO HO HO O OH b O O N N 1 O O HO HO H3CO OCH O OH 3 FIGURE 4.6 Parry pathway for biogenesis of 1. 69, presumably via a facile 2,3-sigmatropic rearrangement of the corresponding N-ylide of ammo- nium salt 68. Dieckman cyclization of diester 69 and subsequent decarboxylation produced the cyclic ketone 65. Pd/Cu catalyzed oxidation furnished the diketone 70, which was cyclized to form the pentacyclic enone 63. On short exposure to zinc dust in hot glacial acetic acid, enone 63 was transformed into the desired pentacyclic ketone 62. This remarkably facile transformation can be rationalized as a transannular reductive rearrangement via a transient bridged aziridinium interme- diate;48 this type of skeletal rearrangement can also be found in Buchi’s synthesis of Iboga alkaloids.49 Subjecting 62 to Moriarity oxidation gave a hydroxy dimethylketal as the sole product,50 which was deduced to be 71 by spectroscopic analysis.51 Deketalization of 71 in THF followed by autoxidative dehydrogenation of the resulting crude hydroxy ketone 72 (epidesmethylcephalotaxine) afforded desmethylcephalotaxinone (73).52 The synthetic product was identical in every respect with a sample prepared from natural (-)-CET by hydrolysis and autoxidative dehydrogenation, as for 71→73. Because desmethylcephalotaxinone (73) has been converted to natural (-)-CET via methylation, optical resolution with L-tartaric acid, and borohydride reduction,53 the sequence outlined in Figure 4.7 KOtBu CO t DMSO-THF 1. (CO2Et) 2E 2 Allyl-Br O O 2. POCl O O 3 DMSO + Br- O N O NH2 3. H O N O N 2, Pd/C CO Et 4. Br(CH2)3CO2Et CO2Et CO2Et 2 66 67 68 69 CO CO2Et 2Et 59% 1. KOtBu,Tol 2. CaCl2,DMSO O Zn/HOAc Pd2+Cu2+ O t N 100oC KO Bu O O O 2, HCl tBuOH DMF-H2O O 65% O N O N O N 78% 82% 62 63 70 65 O O O O 78% O Phl(OAc)2 KOtBu, O KOH, MeOH 2 1. CH(OMe) tBuOH, P(OEt) 3 O O 3 2. resolution O O H N H+/THF 3. NaBH H N N 4 H N O O O 86% O HO HO MeO OMe HO HO OCH O O 3 71 72 73 2 FIGURE 4.7 Total synthesis of cephalotaxine. Copyright © 2005 CRC Press, LLC Zn-Hg O c.HCl reflux O O Wacker Oxi. N N O N or O 74% O Zn/HOAc 100oC O 70-76% 75 65 74 O 70% 1. mCPBA 2. TFAA HOAc, O O 2 (air) O H2, PdC O N Fe2+, 100o N C N O or Zn/HOAc O 57% O 62 100oC 70-78% 77 76 O O O FIGURE 4.8 Enamine cyclopentenone annulation pathway. constitutes a total synthesis of CET with an overall yield of about 12% to 73 from diester 67 through an eight-stage sequence. The synthesis described above evolved from an initial strategic plan (Figure 4.5, plan b) that was based on an intramolecular Mannich cyclization of the iminium intermediate for the E-ring formation.54 The precursor to 64 would be generated from cyclic ketone 65 by a Clemmensen reductive rearrangement. The anomalous Clemmensen reaction of a cyclic α-amino ketone was first noticed by Clemo et al. in 1931,55,56 clarified by Prelog in 1939,57 and later studied systemat- ically by Leonard and coworkers in 1949.58,59 This unique Clemmensen–Clemo–Prelog–Leonard reductive rearrangement was applied to constructing the benzazepine-bearing ABCD-ring system of CET. Clemmensen reduction (Zn-Hg, conc. HCl, reflux) of α-amino ketone 65 led to allyl benzazepine derivative 74 (Figure 4.8). Clemmensen reduction conditions, with zinc dust in hot glacial acetic acid, were also equally effective for this reductive rearrangement.60 This facile rearrangement can be generalized as an acid-catalyzed transannular interaction of nitrogen with the protonated carbonyl group, leading to a transient aziridinium intermediate that would facilitate reductive cleavage of the benzylic C-N bond.61–64 The electron-rich aromatic system certainly contributed to this facile process. Acidic Wacker oxidation of 74 gave methyl ketone 75 (Figure 4.8). However, the attempted generation of iminium intermediate 64 from 75 using Hg(OAc)2 resulted in a slow decomposition of 75 under a variety of reaction conditions.65,66 In the hope that an alternative Polonski–Potier protocol might be more selective, the corresponding amine oxide of 75, prepared by m-CPBA oxidation, was treated with excess trifluoroacetic anhydride in CH2Cl2. Enamine ketone 76 was obtained as the sole product, apparently formed by isomerization of the initially generated iminium salt 64.67 Compound 76 had been previously synthesized by an alternative enamine alkylation of the so-called Dolby–Weinreb enamine.34 It was thought54,68 that cyclization to 62 (formally an endocyclic enamine annulation) could not be realized under various conditions, although Dolby et al.46 reported that acid-catalyzed cyclization of 76 led to a rearranged product (saturated enone 63) in low yield. Although keto enamine 76 appeared not to be a promising intermediate, various acidic treatments of 76 were examined. A hot mixture of 40% acetic acid and 76 in an open flask gradually generated a sole isolatable product, whose structure was verified as the pentacyclic enone 77 (Figure 4.8). Further experimentation improved the yield in this cyclization to 57%. This cyclization appears to be an acid-catalyzed O2-dependent process, reasoned to be an unusual azo-Nazarov-type cyclization initiated by an acid-catalyzed autooxidation. Related acid-catalyzed, oxidative cyclization can be found in Woodward’s synthesis of chlorophyll-a69 and in an oxy-Nazarov cyclization40 in Weinreb’s pioneering CET synthesis.52 Because endocyclic enamine 76 is a direct enamine alkylation product, Copyright © 2005 CRC Press, LLC this unusual cyclization can be regarded as a novel endocyclic

enamine cyclopentenone annulation.70 Such processes could have broad applications to the synthesis of complex polycyclic natural alkaloids and other heterocycles. Catalytic hydrogenation of pentacyclic enone 77 gave the pentacyclic ketone 62 in 76% yield. Interestingly, the action of zinc dust in hot HOAc on 77 smoothly produced ketone 62 without causing skeletal rearrangement (Figure 4.8), which further supports the mechanistic rationale for the transannular reductive rearrangement of enone 63 to pentacyclic ketone 62. In conclusion, a short, practical total synthesis of CET has been developed based on a compu- tationally novel strategy featuring key transannular reductive rearrangements to establish the pen- tacyclic ring skeleton. The efficiency and practicality of the synthesis stem from its brevity (nine stages from diester 67), readily available starting materials, simple reactions, inexpensive reagents, and good overall yield. Finally, this synthesis of CET may be regarded as a biomimetic synthesis71,72 in terms of pentacyclic ring construction.47 Further work on the asymmetric strategy is under way.45 IV. MECHANISM OF ACTION HHT and its analogs are dose- and time-dependent inhibitors of protein synthesis. Effects on DNA may also be important. HHT inhibited 50% of tritiated leucine uptake in Hela cells and globin synthesis in reticulocyte lysates at doses of 0.04 and 0.10 µM, respectively. These doses were significantly lower compared with those necessary for other esters to achieve the same results.73 Degradation of polyribosomes, release of completed protein chains, and inhibition of initiation of protein synthesis were observed. HHT and analogs did not affect mRNA binding to the donor site of the ribosome. The alkaloids blocked poly (U)-directed polyphenylalanine synthesis, peptide bond formation, and enzymatic and nonenzymatic binding of the Phe-tRNA to ribosomes.74 These results indicate that HHT inhibited the elongation phase of translation by preventing substrate binding to the acceptor site on the 60s ribosome subunit and, therefore, blocked aminoacyl-tRNA binding and peptide bond formation. Tujebajeva et al. confirmed these observations in 1989.75 In poly (U)- programmed human placenta 80-S ribosomes, he demonstrated that neither nonenzymatic binding nor eEF-1-dependent Phe-tRNA binding at A and P. sites were hindered by HHT, whereas diphe- nylalanine synthesis and puromycin reactions were strongly inhibited by HHT. Thus, HHT was found to be a selective inhibitor of transpeptidation during the elongation cycle. More recent studies in chronic myeloid leukemia (CML) cells indicated both differentiation and induction of apoptosis as potential downstream mechanisms for HHT effects.76–79 The first studies regarding the biological activity of HHT were investigated simultaneously with structural studies.7–9,11,12,26 Cytokinetic studies showed that HHT cytotoxicity was cell-cycle specific, affecting mostly cells in the G1 and G2 phases, as expected for a drug that primarily inhibits protein synthesis.80 Although this finding suggested a possible role of HHT against slowly growing tumors, clonogenic assay studies demonstrated greater activity against rapidly growing tumors.81 Prolonged drug exposure was required for a maximal antitumor effect in vitro, and in vivo studies showed recovery of protein synthesis within 24 h of injection.74,82–84 These data may be relevant to the interpretation of the results with HHT in solid tumors, which mostly used short infusion and exposure schedules. At dose ranges of 0.5 mg to 4.0 mg/kg, HHT had remarkable in vivo effects on L1210 cells in mice. The inhibition of neoplastic proliferation was measured by mitotic index of leukemic cells and by survival of treated animals, which was 142% of control mice. In mice bearing P388 lymphocytic leukemia, survival prolongation of treated animals was >300% that of nontreated mice and indicated the superior antineoplastic effect of HHT compared with isodoses of three other cephalotaxine alkaloids. In vitro and in vivo studies of HHT and analog esters against L1210 leukemia sublines dem- onstrated a proportional relation between cytotoxicity and protein synthesis inhibition. Intracellular retention of radiolabeled harringtonine was found to correlate with protein synthesis, and the Copyright © 2005 CRC Press, LLC majority of the drug was tightly bound to the microsomal structures. Resistance in two sublines corrrelated with rapid drug egress compared with wild-type lines.85 HHT had moderate activity against CD8F1 mammary carcinoma and marginal effect against B16 intraperitoneal melanoma but was ineffective against Lewis lung carcinoma and human colon, lung, and mammary tumor xenografts in immunodeficient mice.86 It caused extensive necrosis of subcu- taneous colon 38 tumors in mice and appeared to delay tumor growth significantly,87 and it had variable efficacy against different solid tumors and leukemias, with a wide range of cytotoxic effects, mainly directed against rapidly proliferating cells, irrespective of their lymphoid or myeloid origin. Among 80 tumors of 13 histologic subtypes tested, HHT was active in 13 (16%). When it was tested in vitro against 10 human leukemia and lymphoma cell lines,81 it demonstrated a 70-fold difference in growth inhibition between the most sensitive and the most resistant lines. The most sensitive lines were HL-60 (presumed acute promyeocytic) and RPMI-8402 (T-cell acute lympho- cytic); moderately sensitive lines included DND-39A (B-cell lymphoma), ML-2 (acute myeloid), MOLT-3 (T-cell acute lymphocytic), and KG-1 (erythroleukemia); and less sensitive lines were Daudi (B-cell lymphoma), NALL-1 (null cell acute lymphocytic), BALM-2 (B-cell acute lympho- cytic), and DND-41 (T-cell acute lymphocytic). Tumors with high growth fractions appeared to be more sensitive to HHT. In contrast to leukemia cell lines, six human neuroblastoma cell lines were found to be sensitive only after exposure to >300 ng/ml of HHT for 7 d.88 The high HHT concentrations needed to suppress tumor growth (>10,000 ng/mL in four of six cell lines) indicated that doses used in the majority of clinical trials would be inactive against neuroblastoma but might be effective against leukemia. However, in vivo studies showed 53% inhibition of neuroblastoma in mice treated with 3.5 mg/kg HHT intraperitoneally 2 d a week for 3 weeks and 41% inhibition of neuroblastoma in mice treated with 0.7 mg/kg daily for 3 d, followed by 2.1 mg/kg daily for 7 d. Survival of treated mice was prolonged significantly.89 Harringtonine induced monocytic differentiation of HL-60 cells after 7 d of liquid culture; morphologic changes typical of granulocytic or monocytic differentiation were detected in four of eight human acute myeloid leukemia (AML) primary cell cultures performed for days in the absence of growth factors.90 After maturation and proliferation arrest, when cultures were prolonged for 10 d, morphologic features of apoptosis were present in recovered cells (i.e., pyknosis of nuclei, karrhexis, and shrinkage of cytoplasm). These results were supported by Zhou et al., who found that HHT at low concentrations of 2–20 ng/ml induced 28% of HL-60 cells to differentiate to macrophage- like cells.91 Visani et al. (1997)76 reported that HHT exerted more cytotoxicity against CML chronic phase cells, in vitro on semisolid cultures, compared with normal bone marrow at 50 ng/ml (P = 0.02) and 200 ng/mL (P = 0.01). They also demonstrated synergistic effects for HHT combinations with interferon-α (IFN-α), cytarabine, or both. For CML blastic phase cells, only the triple combi- nation was effective. Induction of apoptosis by HHT was dose dependent when tested at 100 ng/mL, 200 ng/mL, and 1000 ng/mL in both chronic phase and normal cells, but no apoptosis was observed for CML blastic phase cells. At 2 × 10−7 mol/L and 10−7 mol/L, harringtonine could induce apoptosis in HL-60 cells after exposure for 4 h.78 In agarose gel electrophoresis, DNA extracted from HL-60 cells treated with harringtonine and HHT showed typical internucleosomal DNA degradation (i.e., DNA ladder, nuclear chromosome segmentation and condensation, and cytoplasmic vacuolation). This HHT effect was concentration and time dependent. Exposure to HHT reversibly inhibited glycoprotein synthesis and stimulated lipid-linked oli- gosaccharide formation in vitro in bladder carcinoma cells, whereas prolonged treatment (>8 h) resulted in a generalized suppression of both glycoprotein biosynthesis and lipid-linked oligosac- charide formation. Kinetic studies indicated that the time course for the decrease in glycoprotein biosynthesis and the accumulation of dolichol-linked oligosaccharide paralleled the HHT-induced decrease in protein synthesis.92 HHT inhibited colony formation in a variety of myeloid (50% inhibition at concentration ranges of 7–12 ng/mL) and lymphocytic (ranges of 4–7 ng/mL) cells, as well as in fresh myeloid leukemic Copyright © 2005 CRC Press, LLC cells from patients (50% inhibition at concentration ranges of 2–25 ng/mL) in a dose-dependent manner. Pulse exposure studies showed 50% inhibition of colony formation of HL.60 cells (10–20 ng/ml) after 45 h of exposure, followed by complete inhibition at 72 h of exposure. Radioactive precursor studies in the HL-60 experiment verified inhibition of protein synthesis by HHT, rather than inhibition of RNA and DNA synthesis.91 In vitro and in vivo studies indicated that although P388 cells resistant to doxorubicin and vincristine also showed cross-resistance to HHT, cytarabine-resistant P388 cell lines showed a surprising collateral sensitivity to HHT.93 These observations have potential implications related to multidrug-resistant (MDR) mechanisms of HHT resistance and to therapeutic strategies including combinations of HHT and MDR-blocking agents, as well as HHT plus cytarabine. The wide differences in apoptotic and cytotoxic effects in different cell lines may be a result of differences in expression of P170 glycoprotein (e.g., in blastic versus chronic phases of CML). Resistance to HHT appears to be MDR related (similarly to anthracyclines, vinca alkaloids, and taxol).94 Cell lines with high MDR expression were 15 times more resistant to HHT effects.95 Two leukemic cell lines (K562-HHT and L1210-HHT), rendered 17-fold and 13-fold, respectively, resistant to HHT, demonstrated cross-resistance to doxorubicine, vincristine, and daunorubicine and had increased expression of the MDRI gene. Expression of MDRI P170 in HHT-resistant sublines of K562 also was increased. These observations may explain the reduced antileukemic activity of HHT in refractory AML and in the accelerated–blastic phases of CML compared with chronic phase CML. To elucidate the mechanism of resistance to HHT in leukemic cells, five sublines of human myeloid leukemia K562 cells, which demonstrated progressive resistance to different concentrations of HHT, were established. These sublines were cross-resistant with daunorubicin, vincristine, etoposide, and mitoxanthrone. Immunofluorescence with monoclonal anti-P-glycoprotein antibody MRK16 and Northern blot analysis demonstrated that resistance to HHT was related to the sequen- tial emergence of MRP gene and MDR-1 gene overexpression. In the highly resistant cell sublines, MDRI gene overexpression predominated.96 Another harringtonine-resistant HL-60 cell line, HR20, showed cross-resistance with HHT, doxorubicine, daunorubicin, vincristine, and colchicines. The growth-doubling time and cell numbers in the G1 phase were increased, and the cell line overex- pressed mdr-1 gene and P-glycoprotein.97 HHT was investigated in vitro in combination with cytotoxic agents to detect potential syner- gistic interactions. With the exception of consistent significant synergistic effects with cytarabine14 and modest synergy with 5-fluorouracil and hexamethylene bisacetamide,97,98 no other synergistic interactions were observed.99 V. MEDICINAL CHEMISTRY OF THE DRUG Different activities among naturally occurring Cephalotaxus alkaloids and the antileukemic esters of cephalotaxine are significant issues for drug development. Smith et al.100 have reviewed struc- ture–activity relationships among Cephalotaxus alkaloids and their derivatives. HHT (1) and harringtonine (3) show comparable levels of activity in P388 and L1210 exper- imental leukemia systems (Table 4.1 and Table 4.2). Insertion of an additional methylene group in the terminal portion of the acyl side chain of 3 has little effect on activity. In contrast, removal of the hydroxyl group from the penultimate carbon of the acyl moiety [to give deoxyharringtonine (5)] reduces activity by about one-half. Shifting this same hydroxyl to give isoharringtonine (4) lowers P-388 activity by nearly an order of magnitude (Table 4.1). As indicated previously, natural (-)-cephalotaxine (2) is devoid of antileukemic activity. The corresponding dimethyl ester (42) and its isomeric acid (42a), provided by hydrolysis of 5, are likewise inactive. Thus, an ester linkage is evidently a required, but not sufficient, structural element for activity. In addition, although 56, the dimethyl ester of the dicarboxylic acid derived from Copyright © 2005 CRC Press, LLC TABLE 4.1 Activity of Selected Cephalotaxus Alkaloids against P-388 Lymphocytic Leukemia Dose Animal weight Survival time, T/C Compound (mg/kg) Survivors difference (T-C) T/C (d) (%) Homoharringtonine (1) 2.00 6/6 –3.8 7.5/9.0 1.00 6/6 –2.8 30.5/9.0 338 0.50 6/6 –1.8 24.5/9.0 272 0.25 6/6 –2.2 22.0/9.0 244 Harringtonine (3) 4.00 2/6 –5.5 5.0/9.0 2.00 6/6 –3.3 18.5/9.0 205 1.00 6/6 –2.3 36.5/9.0 405 0.50 6/6 –1.0 26.5/9.0 294 Isoharringtonine (4) 15.00 6/6 –4.3 9.5/9.0 105 7.50 6/6 –3.0 24.5/9.0 272 3.75 6/6 –2.8 15.5/9.0 172 1.87 6/6 –1.3 13.5/9.0 150 Deoxyharringtonine (5) 4.00 6/6 –3.4 14.0/10.0 140 2.00 6/6 –3.3 18.0/10.0 180 1.00 6/6 –2.4 15.5/10.0 155 0.50 6/6 –1.2 14.5/10.0 145 Note: Data presented are representative of

results from several assays with different samples of each alkaloid. Materials are considered active if the survival time of animals treated (T) with them is ≥ 125% of that of the controls (C) (i.e., T/C ≥ 125%). Values are quoted from ref. 8. harringtonine, has not been assayed in the usual tumor systems (P-388, KB), work with a Hela cell system indicated that this acid has no inhibitory activity when tested as a separate entity.73 Various research groups have prepared numerous cephalotaxine derivatives that incorporate “unnatural” acyl groups or other structural variations. Some seemingly subtle structural alterations abolish activity, whereas certain active esters of cephalotaxine have been prepared whose acyl groups bear no resemblance to those of the natural alkaloids. The following paragraph contains examples of modifications leading to abolished or reduced activity. The partial synthesis of deoxyharringtonine (87) provided a diastereomer (88) differing only in the configuration at C2′ of the acyl side chain.101 Compound 88 appears to be less active than 87, although further testing is needed to establish this point definitively.102 Attaching the “wrong” carboxyl group of the acyl moiety to 2 gives an inactive isomer (85) of deoxyharringtonine. A “rearranged” isomer (5a) of deoxyharringtonine, in which the acyloxy group is shifted from C1 to C3, likewise has little or no antileukemic activity.103 Removal of the hydroxyl function from C2′ of the acyl side chain of deoxyharringtonine (87) produces 106 and thereby abolishes activity. When cephalotaxine was acylated with an acid having a hydroxyl group α to a tertiary carboxyl, although the resulting ester (107) incorporated some steric features of the active esters, it showed no more than marginal activity.104 A structurally diverse series of cephalotaxine esters has been prepared with acyl groups that contain conjugated double bonds, aromatic rings (both benzenoid and heterocyclic), chloro, nitro, hydroxyl, and sulfonate groups (78–107) (Figure 4.9). Esters showing at least marginal activity are listed in Table 4.3; others are inactive.102 It has been noted that some seemingly minor alterations in the acyl moieties of the more potent antileukemic esters of cephalotaxine markedly diminish their activity. Accordingly, the structures of some active esters in Table 4.3 may occasion some surprise. No apparent rationale emerges to provide a logical statement of structure–activity relationships for Copyright © 2005 CRC Press, LLC TABLE 4.2 Activity of Selected Cephalotaxus Alkaloids against P-1210 Lymphocytic Leukemia Dose Weight Survival time, T/C Compound (mg/kg) Survivors difference (T–C) T/C (d) (%) Homoharringtonine (1) 2.00 6/6 –3.0 9.2/9.1 101 1.00 6/6 –1.4 13.0/9.1 142 0.50 6/6 –0.8 11.0/9.1 120 0.25 6/6 –0.5 11.2/9.1 123 Cephalotaxine (2) 220.00 6/6 –1.2 9.8/9.6 102 110.00 6/6 0.2 10.3/9.6 107 55.00 6/6 0.5 9.5/9.6 98 Cephalotaxine acetate (2a) 100.00 6/6 –0.6 9.8/9.6 102 50.00 6/6 0.0 10.2/9.6 106 25.00 6/6 –0.3 10.2/9.6 106 Harringtonine (3) 4.00 1/6 –4.1 0.0/9.1 — 2.00 6/6 –2.4 12.5/9.1 137 1.00 6/6 –1.2 12.3/9.1 135 0.50 6/6 –1.0 12.0/9.1 131 Isoharringtonine (4) 15.00 6/6 –3.5 10.0/9.1 109 7.50 6/6 –1.3 11.5/9.1 126 3.75 6/6 –0.5 11.3/9.1 124 1.87 6/6 –1.1 10.0/9.1 109 Note: Data presented are representative of results from several assays with different samples of each akanoid. Materials are considered active if the survival time of animals treated (T) with them is ≥ 125% of that of the controls (C) (i.e., T/C ≥ 125%). Values are quoted from ref. 8. the series of compounds in Table 4.3. One particularly intriguing observation is that methyl cephal- otaxyl itaconate (83) prepared from the natural (-)-isomer of 2 is active (and exceptionally nontoxic), whereas its optical antipode (84) is inactive. However, until additional representatives of the (+)- cephalotaxine ester series have been prepared and tested, generalizations about their activity will be premature. In terms of dose requirements for effective tumor inhibition, harringtonine and HHT are at least an order of magnitude more active than any of the esters in Table 4.3. This work has delineated a number of structural variations in the acyl moiety that greatly reduce or abolish activity. Indeed, with these examples alone, one might infer that there are rather rigid structural requirements for activity in the harringtonine series. Nevertheless, activity appears in several cephalotaxine esters (e.g., 105, the trichloroethoxycarbonyl derivative) having acyl groups with no apparent relationship to that of harringtonine. It should also be noted that most of the cephalotaxine esters described by Mikolajczak et al. in 1977,102 including 78–107, can be prepared without the severe steric constraints that hamper conversion of cephalotaxine to harringtonine. Obviously, possible variations in the acyl group of harringtonine are endless, and the search for harringtonine analogs with improved properties continues. Except in the case of the “rearranged” ester 5a, structure–activity relationships in the harringto- nine series have not been extended to the cephalotaxine moiety. Many structural variations in the cephalotaxine ring system await physiological investigation, with some of the more obvious vari- ations being (a) transformation of the methylenedioxy group at C18 into dimethoxy, dihydroxy, or other functions; (b) aromatic substitutions at C14 or C17; (c) inversion of the oxygen function at C3 to give an epimeric series of esters; (d) quaternarization of the nitrogen (e.g., to form a methiodide); (e) substitution or oxidation at one or more of the methylene groups — C6, C7, C8, Copyright © 2005 CRC Press, LLC O O O 89, R= O O CCl3 N OMe 99, R= O O O O RO 90, R= S Me OH OMe O 100, R= 91, R= CH2Cl O 2, R= H O O 78, R= OEt 92, R= 101, R= O O O NO2 O 79, R= 93, R= 102, R= OMe O O O O O 80, R= O O CCl 103, R= OEt OMe 3 94, R= O O O O 81, R= 104, R= O OMe 95, R= O O O O O 105, R= O CCl 82, R= 3 OMe O 96, R= O O COOMe 83, R= O COOMe OMe 106, R= O 97, R= N O 84, enantiomer of 83 O OH OH 107, R= 85, R= 98, R= O O COOMe O 86, R= COOMe O O N COOMe O O HO H OH 87, R= O MeO H COOMe O COOMe 5a HO 88, R= O FIGURE 4.9 Various synthetic analogs. Copyright © 2005 CRC Press, LLC TABLE 4.3 Activity of Various Cephalotaxine Esters against P-388 Lymphocytic Leukemia Dose Weight Difference Compound Vehiclea (mg/kg/inj) (T-C) T/C (%) 78 D 20 0.1 135 D 20 –0.2 211 D 13 –0.8 154 T 20 –0.1 129 80 B 80 –0.7 145 B 40 –0.9 134 B 20 –1.0 125 B 10 –1.2 136 D 4.4 –1.5 147 D 1.9 0.5 134 83 D 365 –3.0 198 D 240 –1.4 169 D 160 –0.1 183 D 160 –1.1 167 D 80 –0.1 183 D 40 –1.3 135 86 C 20 0.9 131 87 D 5.9 –0.9 184 A 4 0.0 174 A 2 0.8 126 88 D 9 –2.2 131 A 4 0.5 150 95 D 80 0.9 150 D 40 –1.8 125 D 20 0.9 130 99 A 320 –2.3 136 A 160 1.2 154 A 80 –1.0 138 105 D 320 –1.0 172 D 160 –0.9 162 D 160 –0.3 155 D 80 –0.9 183 D 80 –1.3 160 D 40 –0.4 140 D 40 0.9 183 D 20 –2.7 128 D 20 –1.5 160 D 20 –1.0 195 D 13 –0.5 138 D 8.8 –0.3 170 a A: saline; B: water+alcohol+acetone; C: water+acetone; D: water+alcohol, T: saline+Tween 80. Values are quoted from ref. 100. Copyright © 2005 CRC Press, LLC C10, or C11; and (f) unique skeletal alterations via total synthesis procedures including photo- rearrangements or other approaches. VI. DEVELOPMENT OF THE CLINICAL DRUG FROM THE NATURAL PRODUCTS LEAD As previously noted, HHT (1) is the most promising lead compound among the Cephalotaxus alkaloids and is active against a number of murine tumors, including B26 melanoma, CD8F1 mammary carcinoma, L1210 leukemia, and especially colon 38 tumor and P388 leukemia.105 Its concentration in plant extracts is much higher than those of the other alkaloids, and research with 1 has predominated because of its more ready supply and high in vitro activity.14,105 Even though many other Cephalotaxus alkaloids have been isolated from the Cephalotaxus genus and numerous semisynthetic derivatives prepared, HHT (1) and harringtonine (3) remain the most active com- pounds and have entered clinical trials for the treatment of leukemia, as described in the next section. VII. CLINICAL APPLICATIONS A. CHINESE CLINICAL TRIALS TREATING LEUKEMIAS WITH CEPHALOTAXINE ESTERS HHT has been used in China since 1974 for the treatment of leukemia. Many Chinese patients were treated with mixtures of harringtonine and HHT, using a variety of doses and treatment schedules. The greatest benefit was seen in those patients with AML.105 In one initial study, the mixture was used at a dose of 2 mg daily for 14 d by intramuscular injection, and the dose subsequently was increased to 4 mg daily intravenously for 22–86 d, using a ratio of 2:1 harringto- nine:HHT. This preparation was also evaluated at a dose of 5–6 mg daily for 4–6 d every 2–3 weeks. These studies indicated that the maximum tolerated, therapeutically effective dose was 4 mg daily for 14 d. Major dose-limiting toxic effects were cardiovascular disturbances, including hypotension, ventricular tachycardia, and myelosuppression.14 B. PHASE I STUDIES IN THE UNITED STATES The success of the Chinese studies prompted the initiation of clinical studies in the United States, using highly purified HHT. Initial phase I trials in patients with solid tumors employed an infusion of HHT, ranging from 10 min to 3 h.2 Hypotension accompanied by tachycardia proved to be dose limiting in several trials. Continuous infusion ameliorated this tarchycardia and hypotension, and it was recommended for further trials. On the completion of the phase I clinical trials in patients with solid tumors, clinical studies of HHT leukemia were initiated in 49 adult patients with leukemia, including 36 patients with acute nonlymphocytic leukemia (ANLL). Myelosuppression was the major side effect, and the cardiovascular side effects were minimal.14 C. PHASE II STUDIES IN SOLID TUMORS Ajani et al.106 conducted five phase II trials of HHT in 80 patients with advanced solid tumors including malignant melanoma, sarcoma, head and neck carcinoma, breast carcinoma, and colorec- tal carcinoma. Among the 74 evaluable patients, there were no complete (CR) or partial remissions. Runge-Morris et al.107 treated 18 patients with advanced head and neck squamous cell carcinoma with HHT (4 mg/m2) by continuous infusion daily for 5 d every 4 weeks. Hypotension and myelosuppression were the major severe toxicities; none of the 14 evaluable patients demonstrated an objective response. HHT also was given to 15 patients with recurrent or progressive malignant glioma at a dose of 3-4 mg/m2 by continuous intravenous infusion daily for 5 d every 3–4 weeks. No objective tumor regressions occurred.108 Witte et al.109,110 treated previously untreated patients Copyright © 2005 CRC Press, LLC with advanced colorectal carcinoma and renal carcinoma with HHT. No objective responses were observed. Other phase II studies in solid tumors did not demonstrate antitumor efficacy.111 The above-mentioned phase II studies indicated a lack of efficacy for HHT in solid tumors at the dose schedules used. D. PHASE I–II STUDIES IN PATIENTS WITH ACUTE LEUKEMIA Stewart and Krakoff105 evaluated HHT in a phase I clinical trial using a five-times-daily schedule with both bolus administration and continuous infusion of the drug. Their results were consistent with preclinical laboratory results, showing that the antitumor effects of HHT are more dependent on exposure time than on concentration.81 Kantarjian et al.112 investigated HHT in a lower-dose, longer infusion schedule in patients with refractory-recurrent acute leukemia in an attempt to demonstrate efficacy without hypotensive events. HHT was administered by continuous infusion at a dose of 2.5 mg/m2 daily for 15–21 d to 13 patients and at a dose of 3.0 mg/m2 daily for 15 d to 18 patients. Only one patient (3%) achieved a CR. The authors concluded that this schedule of HHT had low antileukemic efficacy. Feldman et al.113 used HHT, 5 mg/m2, by continuous infusion daily for 9 d in patients with refractory-recurrent acute leukemia or blastic phase CML. CRs were achieved in 7 of 43 patients (16%) with recurrent AML, in 0 of 11 patients with AML that primarily was resistant to anthra- cycline-cytarabine combinations, and in 2 of 3 patients whose

disease was resistant to low-dose cytarabine. Side effects included significant hypotensive events, fluid retention, weight gain (29%), and hyperglycemia (63%).113 Ekert et al.114,115 treated 25 Australian patients with AML using either HT or HHT, and two CRs (8%) were obtained. Sullivan and Leydan116 also reported that an elderly patient with newly diagnosed AML achieved a response to single-agent HT. Chinese investigators have suggested that the maximum tolerated dose in pediatric leukemia patients is 7 mg/m2 per day infused over 10 d.117 Two phase I trials in pediatric leukemia demon- strated that children could tolerate higher doses of HHT. The maximum tolerated dose was 8.5 mg/m2 daily for 10 d, and the dose-limiting toxicity was myalgia.117–119 E. PHASE II STUDIES IN MYELODYSPLASTIC SYNDROME (MDS) Twenty-eight patients with MDS or with MDS that evolved into AML were treated with HHT, 5 mg/m2 daily by continuous infusion for 9 d. Seven patients (25%) achieved a CR. In this study, induction death was high (13 of 28 patients), mainly because of myelosuppression-related infec- tions. Myelosuppression was prolonged and severe, but other toxicities were mild.120 The dose schedule of HHT used in this study of elderly patients was effective but toxic. It was postulated that lower dose schedules of HHT might demonstrate similar efficacy and lower toxicity in patients with MDS.14 F. STUDIES IN ACUTE PROMYELOCYTIC LEUKEMIA (APL) Ye et al. (1988) treated 10 patients with APL with harringtonine, 1–3 mg intravenously over 4–5 h for 1–3 courses, each lasting for 13–81 d.121 Interruptions between courses lasted for 5–11 d. A CR was achieved in seven patients (70%). The major side effects were pancytopenia and bone marrow hypoplasia or aplasia. Feldman et al.122 reported a 60% remission rate in patients with refractory-recurrent APL who received HHT. This result indicates that HHT could be an interesting agent in patients with relapsed APL.123 G. PHASE II STUDIES IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS Results of the lower-dose, longer-exposure schedule of HHT indicated effective antiproliferative activity and an acceptable toxicity profile, which could be potentially useful in a chronic myeloproliferative Copyright © 2005 CRC Press, LLC disorder such as CML.14 O’Brien et al.124 conducted a study in patients with late chronic phase CML. Patients received induction with HHT, 2.5 mg/m2, daily by continuous infusion for 14 d every 4 weeks until complete hematologic response (CHR), followed by maintenance therapy with HHT, 2.5 mg/m2, daily for 7 d every month. Among the 71 patients treated, 72% achieved a CHR, and 31% achieved cytogenetic responses. The investigators concluded that HHT was effective as an initial single-agent therapy in patients with chronic phase CML. VIII. CONCLUSIONS Because of their unique structures and strong antileukemic activity, Cephalotaxus alkaloids have attracted a great deal of attention. Many alkaloids were isolated from natural sources and derived by synthetic methodology. Furthermore, much work will be followed from the viewpoint of medic- inal chemistry. There is clear evidence that HHT (1) and harringtonine (3) have an antileukemic effect, but the dose and duration of administration of the drug can determine the efficacy and toxicity. Combination trials of HHT with other antineoplastic agents are now being investigated, and continuous infusion is recommended for further studies. ACKNOWLEDGMENTS We thank Professor K. Takeya, Professor T. Nagasaka, Dr. Y. Hitotsuyanagi, and Dr. S. Morris- Natschke for their valuable suggestions. Thanks are also due to partial support from National Institutes of Health grant CA17625 (K.H.L.). REFERENCES 1. Huang, C.C. et al., Cytotoxicity and sister chromatid exchanges induced in vitro by six anticancer drugs developed in the People’s Republic of China, J. Natl. Cancer Inst., 71, 841, 1983. 2. Grem, J.L. et al., Cephalotaxine esters: anti-leukemic advance or therapeutic failure? J. Natl. Cancer Inst., 80, 1095, 1988. 3. Paudler, W.W., Kerley, G.I., and McKay, J., The alkaloids of Cephalotaxus drupacea and Cephalotaxus fortunei, J. Org. Chem., 28, 2194, 1963. 4. Perdue, R.E., Spetzman, L.A., and Powell, R.G., Am. Hortic. Mag., 49, 129, 1970. 5. Spencer, G.F., Platner, R.D., and Powell, R.G., Quantitative gas chromatography and gas chromatog- raphy-mass spectrometry of Cephalotaxus alkaloids, J. Chromatogr., 120, 335, 1976. 6. Abraham, D.J., Rosenstein, R.D., and McGandy, E.L., Single crystal X-ray structures of chemother- apeutic agents II, the structure of cephalotaxine methiodide, Tetrahedron Lett., 10, 4085, 1969. 7. Powell, R.G. et al., Structure of cephalotaxine and related alkaloids, Tetrahedron Lett., No. 46, 4081, 1969. 8. Powell, R.G., Weisleder, D., and Smith, C.R., Antitumor alkaloids from Cephalotaxus harringtonia: structure and activity, J. Pharm. Sci., 61, 1227, 1972. 9. Powell, et al., Structures of harringtonine, isoharringtonine, and homoharringtonine, Tetrahedron Lett., 825, 1970. 10. Mikolajczak, K.L., Powell, R.G., and Smith, C.R., Deoxyharringtonine, a new antitumor alkaloid from Cephalotaxus. Structure and synthetic studies, Tetrahedron, 28, 1995, 1972. 11. Powell, R.G. et al., Alkaloids of Cephalotaxus harringtonia var. drupacea. 11-Hydroxycephalotaxine and drupacine, J. Org. Chem., 39, 676, 1974. 12. Powell, R.G. et al., Desmethylcephalotaxine and its correlation with cephalotaxine, Phytochem., 12, 2987, 1973. 13. Cassady, J.M. and Douros, J.D., Anticancer Agents based on Natural Product Models, Medicinal Chemistry, Vol. 16, Academic Press, New York, 1980, 341. Copyright © 2005 CRC Press, LLC 14. Kantarjian, H.M. et al., Homoharringtonine in hematologic and solid malignancies (history, current research, and future directions), Cancer, 92, 1591, 2001. 15. Takano, I. et al., Drupangtonine, a novel antileukemic alkaloid from Cepalotaxus harringtonia var. Drupacea, Bioorg. Med. Chem. Lett., 6, 1689, 1996. 16. Takano, I. et al., Alkaloids from Cephalotaxus harringtonia, Phytochem., 43, 299, 1996. 17. Takano, I. et al., New Cephalotaxus alkaloids from Cephalotaxus harringtonia var. drupacea, J. Nat. Prod., 59, 965, 1996. 18. Takano, I. et al., Cephalotaxidine, a novel dimeric alkaloid from Cepharotaxus harringtonia var. drupacea, Tetrahedron Lett., 37, 7053, 1996. 19. Takano, I. et al., New oxygenated Cephalotaxus alkaloids from Cephalotaxus harringtonia var. drupacea, J. Nat. Prod., 59, 1192, 1996. 20. Takano, I., et al., Ester-type Cephalotaxus alkaloids from Cephalotaxus harringtonia var. drupacea, Phytochem., 44, 735, 1997. 21. Morita, H. et al., Cephalezomines A-F, potent cytotoxic alkaloids from Cephalotaxus harringtonia var. nana, Tetrahedron, 56, 2929, 2000. 22. Bocar, M., Jossang, A., and Bodo, B., New alkaloids from Cephalotaxus fortunei, J. Nat. Prod., 66, 152, 2003. 23. Kobayashi, J. et al., Cephalocyclidin A, a novel pentacyclic alkaloid from Cephalotaxus harringtonia var. nana, J. Org. Chem., 67, 2283, 2002. 24. Powell, R.G., Structures of homoerythrina alkaloids from Cephalotaxus harringtonia, Phytochem., 11, 1467, 1972. 25. Furukawa, H. et al., Alkaloids of Cephalotaxus wilsoniana Hay. in Taiwan, Yakugaku Zasshi, 96, 1373, 1976. 26. Powell, R.G. et al., Alkaloids of Cephalotaxus wilsonia, Phytochem., 11, 3317, 1972. 27. Arora, S.K. et al., Crystal and molecular structure of cephalotaxine p-bromobenzoate, J. Org. Chem., 39, 1269, 1974. 28. Arora, S.K. et al., Crystal and molecular structure of cephalotaxine, J. Org. Chem., 41, 551, 1976. 29. Takano, I. et al., Modification of the skeleton of homoharringtonine through unusual rearrangements, J. Org. Chem., 62, 8251, 1997. 30. Auerbach, J., Ipaktchi, T., and Weinreb, S.M., Synthesis of the diacid side chain of deoxyharringtonine, Tetrahedron Lett., 4561, 1973. 31. Ipaktchi, T. and Weinreb, S.M., Relative configuration of the diacid side chain of isoharringtonine., Tetrahedron Lett., 3895, 1973. 32. Kelly, T. et al., Regiospecific synthesis of the acyl portion of harringtonine, Tetrahedron Lett., 36, 3501, 1973. 33. Utawanit, T., Ph.D. Thesis, University of Illinois at Urbana-Champaign, Urbana Illinois, 1975. 34. Jalil Miah, M.A., Hudlicky, T., and Reed, J.W., in The Alkaloids; Vol. 51, Brossi, A., Ed, Academic Press, New York, 1998, 199. 35. Suga, S., Watanabe, M., and Yoshida, J., Electroauxiliary-assisted sequential introduction of two carbon nucleophiles on the same α-carbon of nitrogen: Application to the synthesis of spiro compounds, J. Amer. Chem. Soc., 124, 14824, 2002. 36. Koseki, Y. et al., A formal total synthesis of (+)-cephalotaxine using sequential N-acylimnium ion reactions, Org. Lett., 43, 6011, 2002. 37. Tietze, L.F. and Schirok, H., Enantioselective highly efficient synthesis of (-)-cephalotaxine using two palladium-catalyzed transformation, J. Am. Chem. Soc., 121, 10264, 1999. 38. Worden, S.M., Mapitse, R., and Hayes, C., Towards a total synthesis of (-)-cephalotaxine: construction of the BCDE-tetracyclic core, Tetrahedron Lett., 43, 6011, 2002. 39. Bocker-Milburn, K.I. et al., Formal intramolecular [5 + 2] photocycloaddition reactions of maleimides: a novel approach to the CDE ring skeleton of (-)-cephalotaxine. Org. Lett., 3, 3005, 2001. 40. Kim, S.H. and Cha, J.K., Synthetic studies toward cephalotaxine: Functionalization of tertiary N- acylhemiaminals by Nazarov cyclization, Synthesis, 2113, 2000. 41. Beall, L.S. and Padwa, A., An approach to the cephalotaxine ring skeleton using an ammonium ylide/steveno[1,2]-rearrangement, Tetrahedron Lett., 39, 4159, 1998. Copyright © 2005 CRC Press, LLC 42. Molander, G.A. and Hierseman, M., Intramolecular 1,3-dipolar cycloaddition as a total for the prep- aration of azaspirocyclic keto aziridines. Synthesis of intermediates for the total synthesis of (±)ceph- alotaxine, Tetrahedron Lett., 38, 4347, 1997. 43. De Oliveira, E.R., Dumas, P., and D’Angela, J., A simple, efficient access to functionalized pyr- rolobenzazapines related to the ABC core of cephalotaxine, Tetrahedron Lett., 38, 3723, 1997. 44. Huang, L. and Xue, Z., in The Alkaloids, Vol. 23, Brossi, A., Ed, Academic Press, New York, 1984, 157. 45. Li, W.D.Z. and Wang, Y.Q., A novel and efficient total synthesis of cephalotaxine, Org. Lett., 5, 2931, 2003. 46. Dolby, L.I., Nelson, S.I., and Senkovich, D., Synthesis of cephalotaxine, J. Org. Chem., 37, 3691, 1972. 47. Parry, R.I. et al., Biosynthesis of the Cephalotaxus alkaloids. Investigations of the early and late stages of cephalotaxine biosynthesis, J. Am. Chem. Soc., 102, 1099, 1980. 48. Nagata, W., Lectures in heterocyclic chemistry, Castle, R.N., Elslager, E.F., Eds., J. Heterocyclic Chem., 1, 529, 1972. 49. Büchi, G. et al., The total synthesis of Iboga alkaloids, J. Am. Chem. Soc., 88, 3099, 1966. 50. Moriarity, R.M., Hu, H., and Gupta, S.C., Direct α-hydroxylation of ketones using iodosobenzene, Tetrahedron Lett., 22, 1283, 1981. 51. Yasuda, S., Yamada, T., and Hanaoka, M., A novel and stereoselective synthesis of (±)-cephalotaxine and its analogue, Tetrahedron Lett., 27, 2023, 1986. 52. Weinreb, S.M. and Auerbach, I., Total synthesis of the Cephalotaxus alkaloids cephalotaxine, ceph- alotaxinone, and demethylcephalotaxinone, J. Am. Chem. Soc., 97, 2503, 1975. 53. Zhong, S. et al. Total synthesis of (-)-cephalotaxine. Zhongguo Yaowu Huaxue Zazhi, 4, 84, 1994. 54. Overman, L.E. and Ricca, D.J., in Comprehensive Organic Synthesis, Vol. 3, Trost, B.M. and Flem- ming, I., Eds., Pergamon Press, Oxford, 1991, 1007. 55. Clemo, G.R. and Ramage, G.R., The lupine alkaloids. Part IV. The synthesis of octahydropyridocoline, J. Chem. Soc., 437, 1931. 56. Clemo, G.R., Raper, R. and Vipond, H.J., The Clemmensen reduction of certain α-amino-ketones and its bearing on the reduction of 1-keto-octahydropyridocoline, J. Chem. Soc., 2095, 1949. 57. Prelog, V. and Seiwerth, R., Constitution of the so-called norlupinane B, Chem. Ber., 72, 1638, 1939. 58. Leonard, N.J. and Ruyle, W.V., Rearrangement of α-aminoketones during Clemmensen reduction. II. Contraction of a six-membered ring in the monocyclic series, J. Am. Chem. Soc., 71, 3094, 1949. 59. Leonard, N.J. and Barthel, E., Jr., Rearrangement of α-aminoketones during Clemmensen reduction. III. Contraction of a seven-membered ring in the monocyclic series, J. Am. Chem. Soc., 71, 3098, 1949. 60. Vedejs, E., Clemmensen reduction of ketones in anhydrous organic solvents, Org. React., 22, 401, 1975. 61. Leonard, N.J., J. Rec. Chem. Prog., 17, 243, 1954. 62. Gaskel, A.J. and Joule, I.A., The zinc-acetic acid reduction of reserpine and other tetrahydro--carbolone alkaloids, Tetrahedron, 24, 5115, 1968. 63. Noe, E. et al., Synthesis of the new (cyclopenta[b]pyrolo[1,2-d]azepine[4,5-b]indole ring system, Tetrahedron Lett., 37, 5701, 1996. 64. Ait-Mohand, S. et al., Rearrangements and cyclizations of 2-chloropropenyl-appended indolo[2,3- a]quinolizidine derivatives, Eur. J. Org. Chem., 3429, 1999. 65. Leonard, N.J. et al., Unsaturated amines. III. Introduction of α,β-unsaturation by means of mercuric acatate:1(10)-dehydroquinolizidine, J. Am. Chem. Soc., 77, 439, 1955. 66. Leonard, N.J., Fulmax, R.W. and Hay, A.S., Unsaturated amines. VII. Introduction of α, β-unsaturation by means of mercuric acetate: methylquinolizidines, J. Am. Chem. Soc., 78, 3457, 1956. 67. Grieson, D.S. and Husson, H.P., in Comprehensive Organic Synthesis; Vol. 6, Trost B.M., Flemming, I. Eds, Pergamon Press, Oxford, 1991, 910. 68. Weinstein, B. and Craig, A.R., Synthetic approach to the cephalotaxine skeleton, J. Org. Chem., 41, 875, 1976. 69. Woodward, R.B. et al., The total synthesis of chlorophyll-a, Tetrahedron, 46, 7599, 1990. 70. Stevens, R.V., General methods of alkaloid synthesis, Acc. Chem. Res., 10, 193, 1977. 71. Marino, J.P. and Samanen, J.M., Biogenetic-type approach to homoerythrina alkaloids, J. Org. Chem., 41, 179, 1976. Copyright © 2005 CRC Press, LLC 72. Kupchan, S.M., Dhingra, O.P.

and Kim, C.K., New biogenetic-type approach to Cephalotaxus alka- loids and the mechanism of Schelhammer-type homoerythrinadienone formation in vitro, J. Org. Chem., 43, 4461, 1978. 73. Huang, M.T., Harringtonine, an inhibitor of initiation of protein biosynthesis, Molec. Pharmacol., 11, 511, 1975. 74. Fresno, M., Jimenez, A., and Vazquez, D., Inhibition of translation in eukaryotic systems by harringto- nine, Eur. J. Biochem., 72, 323, 1977. 75. Tujebajeva, R.M. et al, Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bone formation, FEBS Lett., 257, 254, 1989. 76. Visani, G. et al., Effect of homoharringtonine alone and in combination with alpha interferine and cystosine arabinoside on “in vitro” growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoitic progenitors, Leukemia, 11, 624, 1997. 77. Kuliczkowski, K., Influence of harringtonine on human leukemia cell differentiation, Arch. Immunol. Ther. Exp. (Warsz), 37, 69, 1989. 78. Li, L. et al., Induction of apoptosis by harringtonine and homoharringtonine in HL-60 cells, Yao Hsueh Hsueh Pao, 29, 667, 1994. 79. O’Brien, S., et al., Homoharringtonine induces apoptosis in chronic myelogenous leukemia cells, Blood, 82(Suppl.), 555a. (2203), 1993. 80. Baaske, D.M. and Heinstein, P., Cytotoxicity and cell cycle specificity of homoharringtonine, Anti- microb. Agents Chemother., 12, 298, 1977. 81. Takemura, Y. et al, Biological and pharmacologic effects of harringtonine on human leukemia- lymphoma cells. Cancer Chemother. Pharmacol., 14, 206, 1985. 82. Han, R. and Ji, X.J., Chemical, pharmacological and clinical studies on the antitumor active principle of Cephalotaxus hainanesis Li (in Chinese), Zhonghua Zhongliu Zazhi, 1, 176, 1979. [NIH Library translation (NIH Pub No. 87-2361)]. 83. Wang, Y., Pan, Z., and Han, R., The cytokinetic effects of harringtonine on leukemia L1210 cells, II. Studies by microscopic photometer, Zhonghua Zhognliu Zachi, 2, 247, 1980. 84. Xu, Y. et al., The effect of harringtonine and its allied alkaloids on the incorporation of labeled amino acids into proteins of cells of transplantable leukemias L615 and P388, Acta Pharm. Sinica, 16, 661, 1981. 85. Chou, T.C. et al., Uptake, initial effects, and chemotherapeutic efficacy of harringtonine in murine leukemia cells sensitive and resistant to vincristine and other chemotherapeutic agents, Cancer Res., 43, 3074, 1983. 86. O’Dwyer, P.J. et al., Homoharringtonine perspective on an active new natural product, J. Clin. Oncol., 4, 1563, 1986. 87. Baguley, B.C. et al., Comparison of the effects of flavone acetic acid, fostriecin, homoharringtonine and tumor necrosis factor alpha on colon38 tumours in mice, Eur. J. Cancer Clin. Oncol., 25, 263, 1989. 88. Tebbi, C.K., Chervinsky, D., and Murphy, T., Effects of homoharringtonine on human neuroblastoma cell lines, Proc. Am. Assoc. Cancer Res., 28, 425, 1987. 89. Tebbi, C.K. and Chervinsky, D., The effects of homoharringtonine (HHT) on mouse C-1300 neuro- blastoma in vitro and in vivo, Proc. Am. Assoc. Cancer Res., 27, 274, 1986. 90. Boyd, A.W. and Sullivan, J.R., Leukemic cell differentiation in vivo and in vitro: arrest of proliferation parallels the differentiation induced by the antileukemic drug harringtonine, Blood, 63, 384, 1984. 91. Zhou, J.Y. et al., Effect of homoharringtonine on proliferation and differentiation of human leukemic cells in vitro, Cancer Res., 50, 2031, 1990. 92. Ling, Y.H., Tseng, M.T., and Harty, J.L., Effects of homoharringtonine on protein glycosylation in human bladder carcinoma cell T-24, Cancer Res., 49, 76, 1989. 93. Wilkoff, L.J. et al., Effect of homoharringtonine on the viability of murine leukemia P388 cells resistant to either adriamycin, vincristine, or 1-β-D-arabinofuranosycytosine, Cancer Chemother. Pharmacol., 23, 145, 1989. 94. Tebbi, C.K., Chervinsky, D., and Baker, R.M., Modulation of drug resistance in homoharringtonine- resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole, J. Cell. Physiol., 148, 464, 1991. Copyright © 2005 CRC Press, LLC 95. Russo, D. et al., MDR-related P170-glycoprotein modulates cytotoxic activity of homoharringtonine, Leukemia, 9, 513, 1995. 96. Zhou, D.C. et al., Sequential emergence of MRP-and MDRI-gene over-expression as well as MDRI- gene translocation in homoharringtonine-selected K562 human leukemia cell lines, Int. J. Cancer, 65, 365, 1996. 97. Laster, W.R. et al., Therapeutic synergism (TS) of homoharringtonine (H) plus 5-fluorouracil (FU) against leukemia P388 (P388/o) and ARA-C-resistant P388 (P388/ARA-C), Proc. Am. Assoc. Cancer Res., 23, 199, 1982. 98. Fanucchi, M.P., Kong, X.R., and Chou, T.C., Hexamethylene bisacetamide (HMBA) does not enhance the cytotoxic effects of adriamycin (ADR), 1β-D-arabinofuranosylcytosine (ARA-C) and harringto- nine (HT) in HL-60 cells [abstract 1492], Proc. Am. Assoc. Cancer Res., 27, 376, 1986. 99. Okano, T. et al., Effects of harringtonine in combination with acivicin, adriamycin, L-asparaginase, cytosine arabinoside, dexamethasone, fluorouracil or methotrexate on human acute myelogenous leukemia cell line KG-1, Invest. New Drugs, 1, 145, 1983. 100. Smith, C.R., Jr., Mikolajczak, K.L., and Powell, R.G., Harringtonine and related cephalotaxine esters, in Anticancer Agents Based on Natural Product Models, Cassady, J.M. and Douros, J.D. Eds., Academic Press New York, 1980, pp. 407-414. 101. Mikolajczak, K.L. et al., Synthesis of deoxyharringtonine, Tetrahedron Lett., 283, 1974. 102. Mikolajczak, K.L., Smith, C.R., and Weisleder, D., Synthesis of cephalotaxine esters and correlation of their structures with antitumor activity, J. Med. Chem., 20, 328, 1977. 103. Mikolajczak, K.L., Powell, R.G., and K.L., Smith, C., Preparation and antitumor activity of a rear- ranged ester of cephalotaxine, J. Med. Chem., 18, 63, 1975. 104. Mikolajczak, K.L., Smith, C.R., and Powell, R.G., Partial synthesis of harringtonine analogs, J. Pharm. Sci., 63, 1280, 1974. 105. Stewart, J.A. and Krakoff, I.H., Homoharringtonine: a phase I evaluation, Investigational New Drugs, 3, 279, 1985. 106. Ajani, J.A., et al., Phase II studies of homoharringtonine in patients with advanced malignant melanoma; sarcoma; and head and neck, breast, and colorectal carcinomas., Cancer Treat. Rep., 70, 375, 1986. 107. Runge-Morris, M.A. et al., Evaluation of homoharringtonine efficacy in the treatment of squamous cell carcinoma of the head and neck: A phase II Illinois Cancer Council Study, Invest. New Drugs, 7, 269, 1989. 108. Feun, L.G. et al., Phase II study of homoharringtonine in patients with recurrent primary malignant central nervous system tumors, J. Neurooncol, 9, 159, 1990. 109. Witte, R.S. et al., A phase II trial of amonafide, caracemide, and homoharringtonine in the treatment of patients with advanced renal cell cancer, Invest. New Drugs, 14, 409, 1996. 110. Witte, R.S., A phase II trial of homoharringtonine and caracemide in the treatment of patients with advanced large bowel cancer, Invest. New Drugs, 17, 173, 1999. 111. Kavanagh, J.J. et al., Intermittent IV homoharringtonine for the treatment of refractory epithelial carcinoma of the ovary: a phase II trial, Cancer Treat. Rep., 68, 1503, 1984. 112. Kantarjian, H.M. et al., Phase II study of low-dose continuous infusion homoharringtonine in refractory acute myelogenous leukemia, Cancer, 63, 813, 1989. 113. Feldman, E.J. et al., Homoharringtonine is safe and effective for patients with acute myelogenous leukemia, Leukemia, 6, 1185, 1992. 114. Ekert, H. and Richards, M., Experience with homoharringtonine in one patient with acute myeloid leukemia, Proc. Clin. Oncol. Soc. Aust., 8, 152, 1980. 115. Ekert, H., et al., Treatment of acute myeloid leukemia with the harringtonines. Proc. Clin. Oncol. Soc. Aust., 9, 122, 1982. 116. Sullivan, J. and Leyden, M., Long survival in an elderly patient with acute myeloid leukemia after treatment with harringtonine, Med. J. Aust., 142, 693. 1985. 117. Bell, B.A., Chang M.N., and Weinstein H.J., A Phase II study of homoharringtonine for the treatment of children with refractory or recurrent acute myelogenous leukemia: a pediatric oncology group study, Med. Pediatric. Oncol., 37, 103, 2001. 118. Bell, B.A. et al., Phase II study of homoharringtonine (HHT) for the treatment of children with refractory nonlymphoblastic leukemia (ANLL), Proc. Am. Soc. Clin. Oncol., 13, A1060, 1994. Copyright © 2005 CRC Press, LLC 119. Tan, C.T.C. et al., Phase I trial of homoharringtonine in children with refractory leukemia, Cancer Treat. Rep., 71, 1245, 1987. 120. Feldman, E.J. et al., Homoharringtonine in patients with myelodysplastic syndrome (MDS) and MDS evolving to acute myeloid leukemia, Leukemia, 10, 40, 1996. 121. Ye, J.S. et al., Small-dose harringtonine induces complete remission in patients with acute promye- locytic leukemia, Leukemia, 2, 427, 1988. 122. Feldman, E.J. et al., Acute promyelocytic leukemia: A 5-year experience with new antileukemic agents and a new approach to preventing fatal hemorrhage, Acta Haematol, 82, 117, 1989. 123. Cortes, J. and Kantarjian, H.M., Promising approaches in acute leukemia, Invest. New Drugs, 18, 57, 2000. 124. O’Brien, S. et al., Homoharringtonine therapy induces responses in patients with chronic myelogenous leukemia in late chronic phase, Blood, 86, 3322, 1995. Copyright © 2005 CRC Press, LLC 5 Podophyllotoxins and Analogs1 Kuo-Hsiung Lee and Zhiyan Xiao CONTENTS I. Introduction II. History III. Structure IV. Development of Etoposide and Teniposide V. Mechanisms of Action A. Inhibition of Tubulin Polymerization B. Inhibition of DNA Topoisomerase II C. Other Antineoplastic Mechanisms VI. Structure–Activity Relationships A. Molecular Area-Oriented Analog Syntheses 1. Ring A 2. Ring B 3. Ring C 4. Ring D 5. Ring E B. SAR Models 1. Composite Pharmacophore Model 2. Comparative Molecular Field Analysis Model 3. K-Nearest Neighbor QSAR Model C. Representative Analogs 1. Etopophos 2. NK 611 3. GL-331 4. TOP-53 VII. Syntheses VIII. Clinical Applications IX. Future Perspectives Acknowledgments References I. INTRODUCTION Podophyllotoxins are important natural products in the armamentarium of antineoplastic agents. The biological assessment of podophyllotoxin (1) was followed by discovery of its mode of action and culminated in the synthesis of the anticancer drugs etoposide (2) and teniposide (3). The long journey from podophyllotoxin to etoposide and teniposide illustrates the fascinating development of clinically useful anticancer drugs from natural product prototypes through chemical modification. It is particularly distinctive that structural variation of podophyllotoxin caused a radical change in 1 Antitumor Agents 240. Copyright © 2005 CRC Press, LLC R O O HO O OH O OH O O O O O O O O H3CO OCH3 H3CO OCH3 OH OCH3 2 R = CH3 1 3 R = S STRUCTURES 1–3 the mechanism of action. Today, several new podophyllotoxin analogs have emerged as potential anticancer drugs. Some recent literature contributions have provided comprehensive updates on various aspects of this compound class.1–7 In this chapter, we highlight recent developments and emphasize critical features of these analogs. II. HISTORY The genus Podophyllum (Podophyllaceae), including American P. peltatum L. and Indian or Tibetan P. emodi Wall (syn. P. hexandrum Royle), has been used for centuries for its medicinal properties. Podophyllum plants have long been valued for their cathartic and cholagogic properties (increasing the flow of bile into the intestine) by the indigenous populations of North America and the Himalayas. Podophyllin, the alcoholic extract of Podophyllum rhizome, was listed in the first American Pharmacopoeia (1820), and was later introduced into European Pharmacopoeias. How- ever, it was removed from the U.S. Pharmacopoeia in 1942 because of its undesirable toxicity.3 In the same year, Kaplan demonstrated the curative effects of podophyllin on the benign tumor Condylomata acuminata,8 which rekindled interest in Podophyllum plants and stimulated intensive studies on the action mode and chemical constituents of podophyllin. Podophyllin was first reported in 1946 to show toxic effects against dividing cells in a manner similar to that of colchicine, the classic antimicrotubule agent.9 Later, its major constituent podophyllotoxin was found to inhibit assembly of the mitotic spindle.10 Other podophyllotoxin compounds were also reported to induce cell cycle arrest at mitosis.11 Both podophyllin and podophyllotoxin exhibited destructive effects on mice tumors,12 which raised hopes for their use in the treatment of malignant tumors. However, the zeal in developing podophyllotoxin as cancer chemotherapy was tempered by its unacceptable side effects, particularly gastrointestinal toxicity.5 Although the direct therapeutic application of podophyllotoxin failed, the pioneer work provided a natural prototype and eventually led to the serendipitous discovery of the semisynthetic anticancer drugs etoposide and teniposide. III. STRUCTURE Podophyllotoxin (1), the major constituent of podophyllin, was first isolated in 1880.13 Its correct structure was resolved chemically in 195114 and was later confirmed by total synthesis.15 Similar to other Podophyllum constituents, 1 is an aryltetralinlactone cyclolignan. Its skeleton is a flat, rigid, five-ring system. The methylenedioxy ring A, tetrahydronaphthalene rings B and C, and lactone ring D make a four-ring pseudoplane to which the pendent aryl ring E is attached pseudoaxially at C1 Copyright © 2005 CRC Press, LLC TABLE 5.1 Chemical Structures of Major Podophyllotoxin Analogs R4 R2 R1 O 4 3 A B C 2 D O O 1 O E CH3O 4' OCH3 OR3 R1 R2 R3 R4 Podophyllotoxin OH H CH3 H Deoxypodophyllotoxin H H CH3 H 4′-Demethylpodophyllotoxin OH H H H

4′-Demethylepipodophyllotoxin H OH H H α-Peltatin H H H OH β-Peltatin H H CH3 OH (Table 5.1). The configurations at the four asymmetric centers (C1, C2, C3, and C4) and the highly strained trans-lactone D ring characterize unique structures of this compound class. Podophyllotoxins are widespread in the plant kingdom and are not limited to the Podophyllaceae family. Key chemical structures include podophyllotoxin, deoxypodophyllotoxin, 4′-demethylpodophyllotoxin, 4′-demeth- ylepipodophyllotoxin (DMEP), α-peltatin, β-peltatin, and their corresponding glycosides (Table 5.1). Most clinically relevant antineoplastic podophyllotoxins are DMEPs. IV. DEVELOPMENT OF ETOPOSIDE AND TENIPOSIDE The complex path from Podophyllum plants to podophyllotoxin and eventually to etoposide and teniposide involved more than a century of study and resulted in successful development of clinically useful drugs from natural sources. The investigators themselves described the story retrospectively.16 In the early 1950s, scientists in Sandoz, Ltd. assumed that, in analogy to cardiac glycosides, podophyllotoxin glycosides might exhibit pharmacological profiles superior to those of the agly- cone. This assumption stimulated extensive efforts to acquire both natural and synthetic Podophyl- lum glycosides and led in 1963 to the development and commercialization of SP-G, the condensation product of the crude Podophyllum glycoside fraction with benzaldehyde. A highly active “antile- ukemia factor” was later isolated from SP-G as a minor component (<0.25%). At low doses, this component significantly inhibited cell proliferation in vitro and considerably prolonged the survival time of leukemic mice. It was identified as 4′-O-demethyl-epipodophyllotoxin benzylidene β-D- glucoside (DEPBG, 4) and had the unique structural features of a free phenolic hydroxyl group at C4′ and an epi configuration at C4. Subsequent synthetic efforts to condense 4′-O-demethyl- epipodophyllotoxin glucoside (DEPG, 5) with various aldehydes and ketones led in the late 1960s to the discovery of etoposide (2) and teniposide (3).16 The FDA approved etoposide for the treatment of testicular cancer in 1983, and teniposide was brought into the U.S. market in 1992. These drugs are currently used against a variety of cancers, including small cell lung cancer, testicular cancer, lymphoma, leukemia, and Kaposi’s sarcoma.17 Copyright © 2005 CRC Press, LLC O O HO CH2OH HO HO O O OH O OH O O O O O O O O O H3CO OCH3 H3CO OCH3 OH OH 4 5 STRUCTURES 4–5 V. MECHANISMS OF ACTION Primary molecular mechanisms underlying the antineoplastic activities of podophyllotoxin analogs include preventing the assembly of tubulin into microtubules or inhibiting the catalytic activity of DNA topoisomerase II, although other known and in some cases ambiguous mechanisms are also involved. A. INHIBITION OF TUBULIN POLYMERIZATION As early as 1947, podophyllotoxin was reported to inhibit assembly of the mitotic spindle and induce arrest of the cell cycle at mitosis.10 It was later found to bind tubulin, the fundamental monomeric protein subunit of microtubules, at the same binding site as colchicine. It binds to tubulin as strongly as colchicine does, but the binding is more rapid and is, in contrast to that of colchicine, reversible.18 The microtubule network forms a vital part of the cytoskeleton in eukaryotic cells and plays an important role in mitosis. Podophyllotoxin reversibly binds to tubulin, disturbs the dynamic equilibrium between the assembly and disassembly of microtubules, and eventually causes mitotic arrest. Most Podophyllum compounds, including the acetal products of podophyllotoxin glucosides and peltatins, share this mechanism of action. On treatment with these compounds, cells can still enter mitosis and undergo a normal pro-phase, but the separation of chromosomes is blocked as a result of the inhibition of mitotic spindle formation. Characterizing the cytotoxic properties of these compounds, cells with clumped chromosomes accumulate in metaphase. B. INHIBITION OF DNA TOPOISOMERASE II Unlike podophyllotoxin, etoposide prevents cells from entering mitosis rather than trapping cells in metaphase. Time course analysis with etoposide in tissue culture shows that disappearance of mitoses begins less than 1 h after drug addition.19 This observation implies that the compound acts in late S or G2 phase of the cell cycle, which is the checkpoint for DNA damage or DNA replication, rather than spindle assembly. Etoposide causes few effects on tubulin polymerization. However, fragmentation of DNA in HeLa cells was observed on treatment with etoposide.20 It was not recognized until the 1980s, however, that the ability of etoposide to induce DNA breaks was mediated by DNA topoisomerase II (topo II).21 DNA topoisomerases are ubiquitous enzymes that control the topological state of DNA. Topo II catalyzes the cleavage and religation of double-strand DNA. Etoposide stabilizes the covalent DNA- enzyme cleavable complex, inhibits the catalytic activity of topo II, and induces topo II–mediated Copyright © 2005 CRC Press, LLC DNA breakage. These actions convert the essential enzyme into a cellular poison, trigger cascade reactions, and eventually lead to cell death.22 The topo II inhibition mechanism is shared by etoposide, teniposide, and other therapeutically important DMEPs. The structural preferences of topo II inhibitors over antimicrotubule agents have been roughly identified as (1) 4′-demethylation, (2) 4β-configuration, and (3) 4β-bulky substitution.3 Because of the severe toxic effects of antimicrotubule agents acting on the colchicine-binding site, topo II inhibitory compounds are of more clinical relevance. C. OTHER ANTINEOPLASTIC MECHANISMS Peroxidases or cytochrome P450 can generate catechol, ortho-quinone, and phenoxyradicals from epipodophyllotoxins. These radical species can covalently bind to DNA and cause DNA damage by forming chemical adducts.23 DNA strand cleavage does not occur with etoposide alone; however, DNA cleavage can be induced in the presence of metal ions, such as Cu2+ and Fe3+, or ultraviolet irradiation, and can be inhibited with known radical scavengers.24 These facts support a role of radical formation for DNA cleavage induced by epipodophyllotoxins. Etoposide and GL-331 (Figure 5.1; cf. Sections VI.A.3 and VI.C.3) also induce cancer cell death with signs of apoptosis. Cellular protein tyrosine phosphatase (PTP) activity was increased significantly after GL-331 treatment, and in addition, GL-331-induced internucleosomal cleavage was efficiently prevented by two PTP inhibitors but not by an inhibitor of serine/threonine phos- phatase.25 Abnormal activation of cyclin B-associated CDC 2 kinase has been noted after treatment with etoposide or GL-331 in various cancer cells. GL-331 treatment in NPC-TW01 cells also increased CDC 25A phosphatase activity and facilitated the association of CDC 25A with Raf-1. Apoptotic DNA fragmentation induced by GL-331 was inhibited by treatment with cyclin B1- specific antisense oligonucleotides.26 These results indicate that PTP, CDC2 kinase, and CDC25 phosphatase might be involved in the induced apoptosis. VI. STRUCTURE–ACTIVITY RELATIONSHIPS The impressive antitumor potency and clinical efficacy of 2 and 3 have prompted extensive molec- ular modifications of the podophyllotoxin prototype, and numerous podophyllotoxin analogs have been synthesized and evaluated since the 1950s. As highlights of such global efforts, several synthetic analogs, including Etopophos (Bristol-Myers), NK 611 (Nippon-Kayaku), GL-331 (NPL at UNC), and TOP-53 (Taiho) (Figure 5.1), have been produced as either clinical drugs or novel clinical trial candidates for various cancers. H3C O O HO N H3C O N N O NO HO 2 HO O HN O O O O O O O O O O O O O O O O O H CO OCH H OCH H CO OCH H CO OCH 3 3 3CO 3 3 3 3 3 O O OH OH OH OH ETOPOPHOS P OH GL-331 NK 611 TOP 53 FIGURE 5.1 Representative podophyllotoxin analogs. Copyright © 2005 CRC Press, LLC TABLE 5.2 Molecular Modifications of Podophyllotoxin OH O 4 A B C D O O O E CH3O 4' OCH3 OCH3 Modifications A ring Removal of the methylenedioxy group to give hydroxy, methoxy, or other oxygenated substituents; replacement of the methylenedioxy ring with heteroaromatic ring systems B ring 5-Oxygenation to give hydroxy or alkoxy groups C ring Extensive C4 modifications, including C4 sugar- and nonsugar- (with O-, S-, N- and C-linkages) substituted derivatives; aromatization of C ring D ring Conversion of the γ-lactone to lactam, cyclopentanone, cyclopentane, sulfide, sulfoxide, sulfone, cyclic ether, and homolactone; substitution of the hydrogen at C2 to halogen atoms, methyl or hydroxyl; replacement of C2 to nitrogen E ring Demethylations; oxidation to the O-quinone; esterification of the C4′ hydroxyl A. MOLECULAR AREA-ORIENTED ANALOG SYNTHESES Previous reviews1,6 have thoroughly discussed the extensive molecular modifications of podophyl- lotoxin, which are summarized in Table 5.2. We highlight herein only the chemical modifications most relevant to the structure–activity relationships (SARs). 1. Ring A The A ring has been modified via two different approaches: opening the methylenedioxy bridge and selectively functionalizing the two phenols,27 and replacing the methylenedioxy ring with heteroaromatic ring systems.28 Neither approach has provided derivatives with potent cytotoxicity or topo II inhibitory activity. 2. Ring B B-ring modification is mainly related to two natural products, α-peltatin and β-peltatin (Table 5.1). Similar to podophyllotoxin, these derivatives act as antimitotic agents and interact only weakly with topo II. Many α-peltatin derivatives were synthesized but were much less active than etoposide against topo II and P388 leukemia.29,30 3. Ring C Modification of the C ring has been quite extensive and focused almost exclusively on the C4 position. 4-Epimerization of podophyllotoxin shifts the molecular target from tubulin to topo II,1 which is the clinically relevant target; therefore, most C4 modifications use epipodophyllotoxins. Etoposide and teniposide were developed through C4 modification in the late 1960s.31,32 Sub- sequently, many other sugar substituted derivatives were prepared. Some of the clinically useful Copyright © 2005 CRC Press, LLC TABLE 5.3 Podophyllotoxin Analogs with Nitrogen-Substituted Sugars CH3 O O O R O 1 R2 O O O O H3CO OCH3 OH L1210 No. R1 R2 (Max % T/C) 1 — — 131 2 OH OH 184 6 OH NH2 272 7 NH2 OH 361 8 OH NHMe 113 9 (NK 611) OH NMe2 438 compounds incorporated nitrogen into the sugar moiety to maintain a favorable activity profile and overcome one of etoposide’s main drawbacks — poor water solubility. These compounds increased survival time in mice with leukemia L-1210 (Table 5.3).33 One of these compounds, NK 611 (9), was brought to clinical trials. The preparation of nonglycosidic C4-substituted epipodophyllotoxins began by making various changes to the hydroxyl moiety, including to esters, carbonates, carbamates, ethers, and thioethers.1 The introduction of nitrogen at the 4-position provided 4β-alkylamino,34 arylamino,35 benzylamino,36 and halogenated anilino37 derivatives. In comparison with etoposide, most nitrogen-analogs exhibited superior potency in cellular protein–DNA complex formation and topo II inhibitory assays, as well as comparable cytotoxicity. Table 5.4 lists some of the most active analogs, among which GL-331 (10) was selected for clinical evaluation. Notably, many 4β-nitrogen substituted derivatives retained cytotoxicity against 2-resistant KB variants (KB = human epidermoid carcinoma of the nasopharynx) with decreased cellular uptake of 2, decreased expression of topo II, or overexpression of multidrug- resistant protein (MDR1).38 These results implied that different C4 substituents might play a signif- icant role in the biochemical determinants of cellular drug uptake in 2-resistant cell lines. Another modification leading to potent compounds was alkylation at the 4β-position. Carbon chains containing hydroxyl, amino, or amido groups were introduced.39 Many of the derivatives exhibited potent cytotoxicity and topo II inhibition. Selected data for representative 4β-alkylated analogs are shown in Table 5.5. TOP-53 (16) is currently in Phase I clinical trial. 4. Ring D The trans-fused γ-lactone D ring in etoposide and its derivatives can be converted to the cis-fused lactone (picro form) under basic conditions and can also be extensively metabolized in vivo to open-ring hydroxy acids. All three metabolic species are inactive.1 To overcome the problem of metabolic inactivation, podophyllotoxin derivatives with differently modified D rings, including lactam,40 cyclopentanone,41 cyclopentane,41 sulfide,41 sulfoxide,41 sulfone,41 Copyright © 2005 CRC Press, LLC TABLE 5.4 4β-Arylamino Derivatives of Podophyllotoxin R HN O O O O H3CO OCH3 OH KB cells Topo II % Protein–DNA No. R (IC50 µM) (ID50 µM) Complex Formation Etoposide — 0.2 50 100 10 (GL-331) NO2 0.49 10 323 11 NH2·HCl 0.8 5 330 12 F 0.24 5 213 13 CN 0.64 10 211 14 COOEt 0.84 5 207 15 3,4-O(CH2)2O 0.68 10 279 cyclic ether,42 2-aza,43 homolactone,44 and C2 substituted45,46 derivatives, have been prepared. Many of these derivatives were less active than their D ring intact congeners, especially in topo II inhibition assays. Tetrahydrofuran D ring derivatives were designed to simultaneously eliminate hydrolysis and epimerization of the γ-lactone ring by replacing the lactone carbonyl with a methylene group (Table 5.6). Compounds 20 and 21 were found to be comparable to 2 in inhibiting topo II and causing DNA breakage but inferior to their parent compounds with an intact D ring.42 5.

Ring E 4′-Demethylation increases the etoposide-like activity (e.g., topo II inhibition) and is considered one of the structural fingerprints for topo II inhibitors in this compound class. Extensive E ring modification, including 3′-nitrogen or 3′,4′-dinitrogen substituted, 4′-acyl, 4′-coupled dimeric, and phosphorous-containing derivatives, was performed by the Bristol Myers Company. Many of these derivatives displayed significant cytotoxicity. The most pronounced compound was the 4′-phosphate ester of etoposide (Etopophos).47 This compound is a water-soluble prodrug of etoposide and is currently used clinically by intravenous administration. Introduction of a chlorine atom in the 2′-position was reported to stabilize the resulting compound to C2 epimerization48; however, it significantly decreased the activity in both cytotox- icity and topo II inhibition assays.49 To conclude the molecular area-oriented SAR discussion, Figure 5.2 summarizes the structural features that are critical for the antineoplastic activity of podophyllotoxin analogs.7 Among these features, a 4β-configuration and a 4′-hydroxyl group are considered to be structural determinants for topo II inhibition, the primary mechanism of action of therapeutically useful analogs. Copyright © 2005 CRC Press, LLC TABLE 5.5 4β-Alkylated Derivatives of Podophyllotoxin R1 N .2HCl R2 O O O O H3CO OCH3 OH Topo II Cytotoxicity (ED50 µg/mL)a No. R1 R2 (IC50 µ*M) SBC-3 A-549 HLE G-402 COLO320DM 1 — — 59.2 1.6 2.9 1.5 4.8 14 16 CH3 (CH2)2N(CH3)2 32.5 0.41 0.82 0.23 0.33 2.0 17 CH3 60.9 0.54 1.8 0.36 0.35 3.2 N 18 29.8 0.16 1.0 0.31 0.18 2.1 N 19 33.6 0.28 0.76 0.13 0.25 0.99 N CH3 a SBC-3, small cell lung cancer; A-549, non–small cell lung cancer; HLE, hepatoma; G-402, renal cancer; COLO320DM, colon carcinoma. TABLE 5.6 Tetrahydrofuran D-Ring Derivatives of Podophyllotoxin R O O 20 R = HNC6H5 O 21 R = HNC6H4F CH3O OCH3 OCH3 Topo II Inhibition, % Protein–DNA ID50 (µM) Complex Formation 2 50 100 20 50 139 21 50 125 Copyright © 2005 CRC Press, LLC 4β-Configuration is essential. Various substitution is tolerable. trans-Lactone with 2α, 3β configuration is very important. R O O O O Dioxolane ring is optimal. Free rotation of ring E is required. H3CO OCH3 OR' 4'-Hydroxy is essential with esterification tolerated. FIGURE 5.2 Summary of structural features critical for the antineoplastic activity of podophyllotoxin derivatives. B. SAR MODELS As the number of epipodophyllotoxin derivatives increases, informative SAR must be assembled in a readily usable format. To date, three different approaches have been applied to generate SAR models for this compound class. 1. Composite Pharmacophore Model The first model is the composite pharmacophore model proposed by MacDonald.50 This model was derived from the superimposition of several topo II inhibitors of the epipodophyllotoxin, anthra- cycline, and aminoacridine classes. As shown in Figure 5.3, the model defined three pharmacophoric domains: the DNA intercalating moiety, which is a planar, polycyclic molecular surface (A); the minor groove binding site, which is a pendant, para-hydroxy- or sulfonylamino-phenyl ring (B); and a variable molecular region, which can accommodate considerable structural diversity (C). Although later modifications, particularly various C4 modifications, have produced SAR consistent with this model, the preparation and evaluation of podophenazine derivatives28 failed to support a crucial role for molecular area A in topo II inhibition. This generalized SAR model for diverse topo II inhibitors implied a common interaction site on the enzyme; nevertheless, no experimental evidence validated the existence of such a putative site. 2. Comparative Molecular Field Analysis Model More recently, the comparative molecular field analysis (CoMFA) technique was applied to 102 epipodophyllotoxin derivatives to generate quantitative SAR (QSAR) models.51 The steric and C A H3CO OCH3 X B FIGURE 5.3 MacDonald’s pharmacophore model. Copyright © 2005 CRC Press, LLC electrostatic contour plots of the final CoMFA model indicated that diverse and bulky substitutions could be accommodated at C4, which agreed with MacDonald’s model. The CoMFA model also suggested that the C4 substituent would interact with the DNA minor groove. On the basis of this model, several etoposide analogs bearing minor groove binding moieties at C4 have been synthe- sized.52 Some of these compounds were more active than etoposide in both cytotoxicity and topo II inhibition assays. 3. K-Nearest Neighbor QSAR Model The application of CoMFA is often undermined by poor reproducibility resulting from inconsistent three-dimensional alignment. A k-nearest neighbor quantitative structure activity relationship (kNN- QSAR) method using alignment-free two-dimensional topological descriptors was applied to 157 epipodophyllotoxins.58 QSAR models were generated using molecular connectivity indices (MCI) and molecular operating environment (MOE) descriptors and were characterized by the values of the internal leave-one-out cross-validated R2 (q2) for the training set and external predictive R2 for the test set. As compared with those values obtained with CoMFA method, the kNN QSAR models afforded higher values of q2 and predictive R2. Because of the physicochemical ambiguity of topological descriptors, the kNN-QSAR models cannot direct chemical modifications on specific molecular areas. However, their high predictive ability should guide the rational design of novel derivatives and focused libraries based on the epipodophyllotoxin skeleton, as well as facilitate the search for bioactive structures from large databases. C. REPRESENTATIVE ANALOGS Despite their impressive clinical efficacy, therapeutic application of etoposide and teniposide is often impeded by problems such as poor water solubility, acquired drug resistance, and metabolic inactivation. Several novel podophyllotoxin analogs, including Etopophos (etoposide phosphate), NK 611, GL-331, and TOP-53, have resulted from the wide range of research programs geared to overcome the above problems. 1. Etopophos The poor water solubility of 2 and 3 presents problems in drug administration and formulation. Accordingly, Etopophos, a water-soluble prodrug of etoposide, was developed.47 Etopophos is less toxic and more active than etoposide in in vivo tumor models. It can be efficiently converted in vivo by endogenous phosphatase to the active drug etoposide and exhibits pharmacological and pharmacokinetic profiles similar to those of etoposide. Notably, the prodrug approach increases the in vivo bioavailability from 0.04% to over 50% and also results in more predictable oral bioavail- ability.53 The improved water solubility and bioavailability made Etopophos preferable to etoposide for routine clinical use, and in 1996, the FDA approved its intravenous use. 2. NK 611 To tackle the problem of poor water solubility, another etoposide derivative, NK 611, was devel- oped.33 Introduction of a 2-dimethylamino group led to a 120-fold increase in water solubility. Compared with 2, NK 611 showed similar antitumor activity against human tumor xenografts but more potent topoisomerase II inhibitory effects and cytotoxic activity against various human cancer lines including lung, gastrointestinal, ovarian, testicular, breast, and head and neck cancers and leukemias. Clinical tests of intravenous and oral formulations of NK 611 indicate that it has better bioavailability than etoposide. However, evidence for cross-resistance between etoposide and NK 611 was also found.7 Copyright © 2005 CRC Press, LLC 3. GL-331 GL-331 is a 4β-arylamino analog of etoposide, with the sugar moiety in 2 replaced by a p-nitro anilino group. GL-331 is more active than 2 in causing DNA double-strand breakage and G2-phase arrest.35 It is also more potent against tumor cells both in vitro and in vivo and, remarkably, overcomes multidrug resistance in many cancer cell lines. Formulated GL-331 shows desirable stability and biocompatibility, as well as similar pharmacokinetic profiles, to those of 2. Initial results from phase I clinical trials in non-small and small cell lung, colon, and head and neck cancers showed marked antitumor efficacy. Side effects were minimal, with cytopenias being the major toxicity. GL-331 is not active against stomach cancer. Phase II clinical evaluation against several forms of cancer, especially etoposide-resistant malignancies, is being planned.54 4. TOP-53 TOP-53 was selected from a series of 4β-alkylated etoposide analogs in which the glycoside group of etoposide was replaced with a carbon chain containing hydroxyl, amino, or amido groups.39 As compared with 2, TOP-53 was a more potent inhibitor of topo II. It showed nearly wild-type potency against a mutant yeast type II enzyme highly resistant to 2, implying therapeutic potential for drug- resistant cancers. TOP-53 exhibited strong activity against a wide variety of tumor cells, with especially high activity against non-small cell lung cancer in both tumor cells and animal tumor models.55 This compound is currently in phase I clinical trials.7 VII. SYNTHESES Gensler et al. accomplished the first total synthesis of podophyllotoxin in the 1960s.15 Major chal- lenges for total syntheses of podophyllotoxin analogs include the presence of four contiguous stere- ocenters and a base-sensitive trans-lactone ring. To date, four general approaches to the syntheses of podophyllotoxin derivatives have been developed. Key steps in these routes involve the elaboration of an χ-oxo ester, the lactonization of a dihydroxy acid, the cyclization of a conjugate addition product, or the use of a Diels–Alder reaction to construct the aryltetralin unit.5 Although several synthetic approaches modified from the above routes provided excellent enantiopurities, the low overall yields disqualified total syntheses as an alternative for naturally produced materials. Therefore, natural (–)-podophyllotoxin remains the major source for the preparation of clinically useful analogs. DMEP was synthesized from podophyllotoxin in the late 1960s via simultaneous 4-epimerization and selective 4′-demethylation using hydrogen bromide.56 After 4′-benzyloxycarbonyl protection to give intermediate 22, the 4β-hydroxyl group was then glycosylated by reaction with tetra-O-acetyl- α-D-glucopyranosyl bromide and subsequent ZnCl2-catalyzed methanolysis.31 The resulting glyco- sidic derivative 23 was treated with aldehydes or ketones in the presence of acid catalysts to yield the corresponding cyclic acetals or ketals, including etoposide and teniposide (Scheme 5.1).32 An efficient preparation of Etopophos is shown in Scheme 5.2.47 The benzyl phosphate pro- tection of the 4′-phenol and glycosylation of C4 with an acetylated sugar characterize this prepa- ration. The overall yield (starting from podophyllotoxin) was over 19%. The analog NK 611 was prepared from the aminosugar intermediate 24, which was itself prepared following the procedure used to synthesize etoposide and teniposide (cf. Scheme 5.1). NK 611 was then produced by reductive methylation with aqueous formaldehyde in the presence of NaBH3CN (Scheme 5.3).33 In sharp contrast to the preparation of the other clinically useful derivatives, a two-step, one- pot procedure was employed in the manufacture of GL-331. GL-331 was obtained from podophyl- lotoxin through subsequent 4-bromination and 4′-demethylation with hydrogen bromide (HBr, g) and nucleophilic displacement with the appropriate amine in the presence of BaCO3 (Scheme 5.4).35 The preparation of the 4β-alkyl derivative TOP-53 is shown in Scheme 5.5.39 The 4′-hydroxyl of DMEP was protected by a benzyloxycarbonyl group before regio- and stereospecific introduction Copyright © 2005 CRC Press, LLC OH OH OH O O O O 1) HBr (g) O CBZ-Cl O O O O O 2) Acetone, H2O O O BaCO3 H3CO OCH3 H CO OCH 3 3 H3CO OCH3 OCH3 OH OCBZ DMEP 22 AcO HO R AcO O O HO O AcO HO O HO O AcO O HO O HO O O O O O O O O H + 3O O RCHO O O O O H3CO OCH3 H C OCH 3 O 3 H OCH 3CO 3 OCBZ OH OH 23 Etoposide (R = CH3) Teniposide (R = C4H3S) SCHEME 5.1 Synthesis of etoposide and teniposide. H3C O H3C O O O O HO O OH OH RO RO O HO O O O H O O 3C O O O O (BzO) O O O 2POCl OH O O RO OR O Zn, AcOH O O CH3CN, O BF3. Et2O O THF O EtN(i-Pr)2 1,2-DCE / -20oC DMAP H3CO OCH H CO OCH 3 3 3 R = CO2CH2CCl H CO OCH H 3 3 3CO OCH 3 3 OH O O O BzO P O BzO P O HO P BzO BzO HO O Etopophos SCHEME 5.2 Synthesis of Etopophos. of allyl at the 4β-position, using trimethylallylsilane in the presence of boron trifluoride etherate. Oxidation of the 4β-allyl derivative (25) with osmic acid and N-oxide (NMO), followed by lead tetraacetic acid, gave the 4β-formylmethyl derivative (26). Reductive amination of 26 gave the 4β- aminoalkyl derivative, and subsequent 4′-deprotection provided the final product TOP-53. H3C O O O H3C O HO HO H2N O N O O O O aq. HCHO, NaBH3CN O O MeCN O O O H3CO OCH3 H3CO OCH3 OH OH 24 NK-611 SCHEME 5.3 Synthesis of NK 611. Copyright © 2005 CRC Press, LLC NO2 OH HN O O O O O 1) HBr (g) O O 2) BaCO O 3 H2N NO2 H O H 3CO C 3 H CH 3CO O 3 OCH3 OH GL-331 SCHEME 5.4 Synthesis

of GL-331. VIII. CLINICAL APPLICATIONS Etoposide shows broad-spectrum antitumor activity against a variety of cancers. It is effective against ascitic tumors, testicular tumors, ovarian and gestational carcinomas, different types of lung cancer (small cell, squamous, adenocarcinoma, Lewis carcinoma), leukemias (monocytic and refrac- tory), malignant and recurrent lymphomas, urogenital tumors, sarcomas, melanomas, and experi- mentally induced colon cancer.6 Etoposide is widely used as an antineoplastic agent, especially in combination chemotherapeutic regimens. The combination of etoposide with cis-platin is a most effective protocol and shows highly productive first-line therapeutic synergism against small cell cancer and as a second-line or salvage therapy for CAV (cyclophosphamide, Adriamycin [doxorubicin], and vincristine)–resistant or CAV-failure small cell lung cancer. It is also effective for the treatment of testicular cancer and non–small cell lung cancer. When incorporated into other multidrug treatment protocols, etoposide is also used for the treatment of non-Hodgkin lymphomas resistant to other agents, different types of lymphomas, refractory childhood leukemia, hepatocellular metastasis, refractory acute lympho- blastic leukemia, and other types of cancers.6,57 As a water-soluble prodrug of etoposide, Etopophos exhibits pharmacological and pharmaco- kinetic profiles similar to those of etoposide and is preferable for routine clinical use, especially for intravenous administration. Teniposide, which is more potent than etoposide, shows good antineoplastic activity against different types of cancer, including lymphoblastic, acute lymphocytic leukemia and other experi- mentally induced leukemias, infantile non-Hodgkin lymphoblastic lymphoma, multiple myeloma, ascitic tumors, malignant brain tumors, colorectal and refractory or recurrent testicular carcinomas, and small cell and non–small cell lung cancer. Combination chemotherapy incorporating teniposide has also been applied to various cancers. It is used in combination with cis-platin against neuro- blastoma, with cytarabine (ara-C), against acute lymphoblastic leukemia, and with carboplatin against small cell lung cancer.6,57 N N OH CH2CH=CH2 CH2CHO O O 1) cat. OsO - O O O 4 1) NaCNBH3 CH NMO/acetone O 2=CHCH2Si O AcOH-CH3OH O O Me3O O O O BF3.Et2O O 2) Pb(OAc)4/ H O O CH2Cl2; -20 - 0 oC benzene N CH H N 3 3C CH3 H C H 2) 10% Pd-C,H2 3 CH 3CO O H H CO OCH 3 3 3CO OCH3 H3CO O 3 OCBZ OCBZ OCBZ (1 atm)/CH2Cl2 OH 25 26 TOP-53 SCHEME 5.5 The synthesis of TOP-53. Copyright © 2005 CRC Press, LLC IX. FUTURE PERSPECTIVES Etoposide and teniposide have been used in cancer chemotherapy for over two decades. However, their clinical application is often impeded by problems of acquired drug resistance and poor water solubility. The recent development of anticancer candidates, including NK 611, GL-331, and TOP- 53, indicates that molecular modification at position 4 of etoposide can effectively yield novel therapeutic analogs with better pharmacological and pharmacokinetic profiles. The availability of reliable and interpretable QSAR models will significantly benefit continued chemical efforts to produce improved analogs. Readily usable QSAR information must be extracted from accumulated structural and biological data. Although antineoplastic properties are arguably the most pronounced pharmacological effect of podophyllotoxin analogs, their antiviral, anti-inflammatory, and immunosuppressive activities are drawing more and more attention. We anticipate podophyllotoxin analogs with therapeutic utility other than cancer chemotherapy to be produced by exploring other biological activities. ACKNOWLEDGMENTS The research on podophyllotoxin analogs performed in the Natural Products Laboratory UNC- Chapel Hill was supported by National Institutes of Health grant CA 17625, awarded to K. H. Lee. REFERENCES 1. Zhang, Y.L. and Lee, K.H., Recent progress in the development of novel antitumor etoposide analogs, Chin. Pharm. J., 46, 319, 1994. 2. Bohlin, L. and Rosén, B. Podophyllotoxin derivatives: drug discovery and development, Drug Dis- covery Today, 8, 343, 1996. 3. Imbert, T.F., Discovery of podophyllotoxins, Biochimie, 80, 207, 1998. 4. Damayanthi, Y. and Lown, J.W. Podophyllotoxins: Current status and recent developments. Curr. Med. Chem., 5, 205, 1998. 5. Canel, C. et al., Podophyllotoxin, Phytochemistry, 54, 115, 2000. 6. Gordaliza, M. et al., Antitumor properties of podophyllotoxin and related compounds, Curr. Pharm. Design, 6, 1811, 2000. 7. Moraes, R.M., Dayan, F.E., and Canel, C., The lignans of Podophyllum, Studies in Natural Products Chemistry, 26 (Bioactive Natural Products [Part G]), 149, 2002. 8. Kaplan I.W., Condylomata acuminata, New Orleans Med. Surg. J., 94, 388, 1942. 9. King, L.S. and Sullivan, M., The similarity of the effect of podophyllin and colchicine and their uses in the treatment of Condylomata acuminata, Science, 104, 244, 1946. 10. Sullivan, B.J. and Weshler, H.I., The cytological effects of podophyllotoxin, Science, 105, 433, 1947. 11. Seidlova-Massinova, V., Malinsky, J., and Santavy, F., The biological effect of some podophyllotoxin compounds and their dependence on chemical structure, J. Natl. Cancer Inst., 18, 359, 1957. 12. Hartwell, J.L. and Shear, M.J., Chemotherapy of cancer: classes of compounds under investigation, and active components of podophyllin. Cancer Res., 7, 716, 1947. 13. Podwyssotzki, V., Pharmakologische studien über Podophyllum peltatum, Arch. Exp. Pathol. Phar- makol., 13, 29, 1880. 14. Hartwell, J.L. and Schrecker, A.W., Components of podophyllin. V. The constitution of podophyllo- toxin, J. Am. Chem. Soc., 73, 2909, 1951. 15. Gensler, W.J. and Gatsonis C.D., Synthesis of podophyllotoxin, J. Am. Chem. Soc., 84, 1748, 1962. 16. Stähelin, H. and von Wartburg, A., The chemical and biological route from podophyllotoxin glucoside to etoposide: ninth Cain memorial award lecture, Cancer Res., 51, 5, 1991. 17. O’Dwyer, P.J. et al., Etoposide (VP-16-213): current status of an anticancer drug, N. Engl. J. Med., 312, 692, 1985. Copyright © 2005 CRC Press, LLC 18. Cortese, F., Bhattacharyya, B., and Wolf, J., Podophyllotoxin as a probe for the colchicine binding site of tubulin, J. Biol. Chem., 252, 1134, 1977. 19. Stähelin, H.F., 4-Demethyl-epipodophyllotoxin thenylidene glucoside (VM-26), a podophyllum com- pound with a new mechanism of action, Eur. J. Cancer, 6, 303, 1970. 20. Loike, J.D. and Horwitz, S.B., Effect of VP-16-213 on the intracellular degradation of DNA in HeLa cells, Biochemistry, 15, 5443, 1976. 21. Ross, W. et al., Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage, Cancer Res., 44, 5857, 1984. 22. Berger J.M. and Wang J.C., Recent developments in DNA topoisomerase II structure and mechanism, Curr. Opin. Struct. Biol., 6, 84, 1996. 23. van Maanen, J.M.S. et al., Effects of the ortho-quinone and catechol of the antitumor drug VP-16- 213 on the biological activity of single-stranded and double stranded ΦX174 DNA, Biochem. Phar- macol., 37, 3579, 1988. 24. Sakurai, H. et al., Metal- and photo-induced cleavage of DNA by podophyllotoxin, etoposide, and their related compounds, Mol. Pharmacol., 40, 965, 1991. 25. Huang, T.S. et al., Protein tyrosine phosphatase activities are involved in apoptotic cancer cell death induced by GL-331, a new homolog of etoposide, Cancer Lett., 110, 77, 1996. 26. Huang, T.S. et al., Activation of CDC 25 phosphatase and CDC2 kinase involved in GL-331- induced apoptosis, Cancer Res., 57, 2974, 1997. 27. Wang, Z.Q. et al., Antitumor agents 124. New 4β-substituted aniline derivatives of 6,7-O,O-demeth- ylpodophyllotoxin and related compounds as potent inhibitors of human DNA topoisomerase II, J. Med. Chem., 35, 871, 1992. 28. Cho, S.J. et al., Antitumor agents 164. Podophenazine, 2″,3″-dichloropodophenazine, ben- zopodophenazine, and their 4β-p-nitroaniline derivatives as novel DNA topoisomerase II inhibitors, J. Med. Chem., 39, 1396, 1996. 29. Thurston, L.S. et al., Antitumor agents 78. Inhibition of human DNA topoisomerase II by podophyl- lotoxin and α-peltatin analogues, J. Med. Chem., 29, 1547, 1986. 30. Saito, H. et al., Studies on lignan lactone antitumor agents IV. Synthesis of glycosidic lignan variants related to α-peltatin, Bull. Chem. Soc. Jpn., 61, 1259, 1988. 31. Kuhn, M. and von Wartburg, A., On a new glycoside synthesis process II. Glycosides of 4′-demeth- ylepipodophyllotoxins, Helv. Chem. Acta, 52, 948, 1969. 32. Keller-Juslen, C. et al., Synthesis and antimitotic activity of glycosidic lignan derivatives related to podophyllotoxin, J. Med. Chem., 14, 936, 1971. 33. Saito, H. et al., Studies on lignan lactone antitumor agents. II. Synthesis of N-alkylamino- and 2,6- dideoxy-2-aminoglycosidic lignan variants related to podophyllotoxin, Chem. Pharm. Bull., 34, 3741, 1986. 34. Lee, K.H. et al., Antitumor agents 107. New cytotoxic 4-alkylamino analogues of 4′-demethylepi- podophyllotoxin as inhibitors of human DNA topoisomerase II, J. Nat. Prod., 52, 606, 1989. 35. Wang, Z.Q. et al., Antitumor agents 113. New 4β-arylamino derivatives of 4′-O-demethylepipodo- phyllotoxin and related compounds as potent inhibitors of human DNA topoisomerase II, J. Med. Chem., 33, 2660, 1990. 36. Zhou, X.M. et al., Antitumor agents 120. New 4-substituted benzylamine and benzyl ether derivatives of 4′-O-demethylepipodophyllotoxin as potent inhibitors of human DNA topoisomerase II, J. Med. Chem., 34, 3346, 1991. 37. Lee, K.H. et al., Antitumor agents 111. New 4-hydroxylated and 4-halogenated anilino derivatives of 4-demethylepipodophyllotoxin as potent inhibitors of human DNA topoisomerase II, J. Med. Chem., 33, 1364, 1990. 38. Chang, J.Y. et al., Effect of 4β-arylamino derivatives of 4′-O-demethylpodophyllotoxin on human DNA topoisomerase II, tubulin polymerization, KB cells, and their resistant variants, Cancer Res., 51, 1755, 1991. 39. Terada, T. et al., Antitumor agents 3. Synthesis and biological activity of 4β-alkyl derivatives con- taining hydroxyl, amino, and amido groups of 4′-O-demethyl-4-deoxypodophyllotoxin as antitumor agents, J. Med. Chem., 36, 1689, 1993. 40. Kadow, J.F., Vyas, D.M., and Doyle, T.W., Synthesis of etoposide lactam via a Mitsunobu reaction sequence, Tetra. Lett., 30, 3299, 1989. Copyright © 2005 CRC Press, LLC 41. Gensler, W.J., Murthy, C.D., and Trammell, M.H., Nonenolizable podophyllotoxin derivatives, J. Med. Chem., 20, 635, 1977. 42. Zhou, X.M. et al., Antitumor agents 144. New γ-lactone ring-modified arylamino etoposide analogs as inhibitors of human DNA topoisomerase II, J. Med. Chem., 37, 287, 1994. 43. Tomioka, K., Kubota, Y., and Koga, K., Synthesis and antitumor activity of podophyllotoxin aza- analogues, Tetra. Lett., 30, 2953, 1989. 44. Roulland, E. et al., Synthesis of picropodophyllotoxin homolactone, Tetra. Lett., 41, 6769, 2000. 45. Glinski-Oomen, M.B., Freed, J.C., and Drust, T., Preparation of 2-substituted podophyllotoxin deriv- atives, J. Org. Chem., 52, 2749, 1987. 46. VanVliet, D.S. et al., Antitumor agents: 207. Design, synthesis, and biological testing of 4β−anilino- 2-fluoro-4′-demethylpodophyllotoxin analogues as cytotoxic and antiviral agents, J. Med. Chem., 44, 1422, 2001. 47. Saulnier, M.G. et al., Synthesis of etoposide phosphate, BMY-40481: a water-soluble clinically active prodrug of etoposide, Bioorg. Med. Chem. Lett., 4, 2567, 1994. 48. Ayres, D.C. and Lim, C.K., Lignans and related phenols. Part XIII. Halogenated derivatives of podophyllotoxin, J. Chem. Soc., Perkin I, 111, 1350, 1972. 49. Hu, H. et al., Antitumor agents 123. Synthesis and human DNA topoisomerase II inhibitory activity of 2′-chloro derivatives of etoposide and 4β-(arylamino)-4′-O-demethylpodophyllotoxins, J. Med. Chem., 35, 866, 1992. 50. MacDonald, T.L. et al., On the mechanism of interaction of DNA topoisomerase II with chemother- apeutic agents. In DNA Topoisomerase in Cancer, Potmesil, M., Kohn, K.W., Eds., Oxford University Press, New York, 1991, 119. 51. Cho, S.J. et al., Antitumor agents 163. Three-dimensional quantitative structure-activity relationship study of 4′-O-demethylepipodophyllotoxin analogs using the modified CoMFA/q2-GRS approach, J. Med. Chem., 39, 1383, 1996. 52. Ji, Z. et al., Antitumor agents 177. Design, syntheses, and biological evaluation of novel etoposide analogs bearing pyrrolecarboxamidino group as DNA topoisomerase II inhibitors, Bioorg. Med. Chem. Lett., 7, 607, 1997. 53. Hande, K.R., Etoposide: Four decades of development of a topoisomerase II inhibitor, Eur. J. Cancer, 34, 1514, 1998. 54. Lee, K.H., Antitumor agents 197. Novel antitumor agents from higher plants, Med. Res. Rev., 19, 569, 1999. 55. Byl, J.A.W., DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action, Biochemistry, 40, 712, 2001. 56. Kuhn, M., Keller-Juslen, C., and von Wartburg, A., Partial syntheses von 4′-demethylepipodophyllo- toxins, Helv. Chem. Acta, 52, 944, 1969 57. Ayres, D.C. and Loike J.D., Lignans. Chemical, biological and clinical properties, Cambridge Uni- versity Press, Cambridge, 1990, 113. 58. Xiao, Z. et al., Antitumor agents 213. Modeling of epipodophyllotoxin derivatives using variable selection k Nearest Neighbor QSAR method. J. Med. Chem., 45, 2294, 2002. Copyright © 2005 CRC Press, LLC 6 Taxol and Its Analogs David G. I. Kingston CONTENTS I. Introduction II. History A. Discovery B. Preclinical Development C. Clinical Development D. The Taxol Supply Crisis E. Taxotere III. Biosynthesis and Bioproduction of Taxol A. Taxol Biosynthesis 1. The Diterpenoid Ring System 2. The Side Chain B. Taxol Bioproduction IV. Taxol’s Mechanism of Action V. Medicinal Chemistry of Taxol A. Ring A Modifications B. Ring B Modifications C. Ring C Modifications D. Ring D Modifications E. Side Chain Analogs F. Prodrugs of Taxol G. Targeted Analogs of Taxol H. Summary of Taxol’s SAR VI. Synthetic Studies A. Semisynthetic Methods B. Total Synthesis VII. The Taxol–Tubulin Interaction VIII. Other Natural Products with a Similar

Mechanism of Action IX. Clinical Applications of Taxol and Docetaxel X. Taxol Analogs in Clinical Trials XI. Conclusion Acknowledgments References I. INTRODUCTION Arguably no naturally occurring anticancer agent has had a bigger effect on cancer treatment than Taxol® (I.1), now known as paclitaxel. Although it is now recognized as one of the most important drugs available for the treatment of breast and ovarian cancers, and although it has Copyright © 2005 CRC Press, LLC AcO O OH O Ph NH O Ph O H O OH OH AcO OCOPh I.1 SCHEME 6.1 spawned several analogs that are now in clinical trials, taxol itself almost never became a drug at all. This review briefly covers the early history of the discovery and development of taxol and then goes on to describe its medicinal chemistry, its synthesis, its interaction with tubulin, and its relationship to compounds such as the epothilones and discodermolide, which have similar mechanisms of action. II. HISTORY A. DISCOVERY The story of taxol began on August 21, 1962, when a team of botanists led by Dr. Arthur Barclay from the U.S. Department of Agriculture, working under a contract from the National Cancer Institute (NCI), collected a sample of Taxus brevifolia Nutt. in the Gifford Pinchot National Forest in Washington state. As was the procedure at that time, the sample was extracted by the Wisconsin Alumni Research Foundation contract laboratory, and the extract was tested for cytotoxicity to KB (human epidermoid carcinoma of the nasopharynx) cells by Microbial Associates in Bethesda, Maryland. A positive response to the stem and bark extract led to the assignment of the extract to Dr. Monroe Wall, who had recently moved to the newly established Research Triangle Institute in North Carolina. Work in the Wall laboratory was also carried out under a contract from the NCI, but it proceeded slowly at first. This was in part because of the complexity of the structural problem and in part because Dr. Wall also had another exciting compound, camptothecin, under investigation, and this study consumed much of his resources. It was thus not until 1971 that Dr. Wall, together with his collaborators Dr. Mansukh Wani and Dr. Andrew McPhail, announced the structure of the major active constituent of T. brevifolia as taxol (I.1).1 Unknown to the discoverers, the name taxol had been trademarked by a French company for an unrelated laxative product. This trademark was later acquired by Bristol-Myers Squibb (BMS), who then applied it to their formulation of the drug. The generic name paclitaxel was assigned to the chemical compound of structure I.1 (Scheme 6.1). Because this review is partly historical in nature, the name taxol is retained for compound I.1. No infringement of the BMS trademark is implied. Interestingly, it has been pointed out2 that the selection of T. brevifolia as the source of taxol was very fortunate, as this species only contains low amounts of the toxic alkaloids taxine A and B. Had a different yew species been investigated, it is probable that Dr. Wall’s fractionation would have led to the isolation of the taxines as the cytotoxic constituents, and the smaller amounts of taxol present in these other species might have gone undetected. B. PRECLINICAL DEVELOPMENT The publication of taxol’s structure and activity excited interest in the natural products community, but not in the pharmaceutical industry. The reasons for this are not hard to discern, as taxol presented a number of seemingly intractable problems to a would-be developer. In the first place it occurred only in low yield in the bark of T. brevifolia, which was its best source, and this bark was relatively thin; it would thus take a Herculean effort to obtain enough taxol for preclinical studies, let alone Copyright © 2005 CRC Press, LLC Heterodimer Initiation Polymerisation/ Formation Elongation α-Tubulin (αβ) β-Tubulin Taxol Stabilized microtubule 12 protofilaments 22nmdiameter FIGURE 6.1 Tubulin polymerization by taxol. clinical trials and clinical use. Complicating this situation was the fact that taxol’s structure was very complex, so it could not be synthesized readily, and it was only very sparingly soluble in water, so its formulation would be difficult. Finally, it only showed activity against KB cell culture and various leukemias and one carcinosarcoma, so it was not clear that it would be a successful drug against solid tumors. Given these handicaps, it is fortunate that taxol became more than a laboratory curiosity. That it did so is in no small part a result of the perseverance of Dr. Wall and Dr. Mathew Suffness at the NCI. Modest amounts of taxol were isolated in the early 1970s to allow for additional animal testing, and a turning point was reached when it was found that taxol showed excellent activity against various human solid tumor xenografts in nude mice, including the CX-1 colon and MX-1 breast xenografts. These results were encouraging enough that in 1977 the NCI decided to begin preclinical formulation and toxicity studies of taxol, with a view to eventual clinical trials. A second major turning point in taxol’s development was the discovery by Dr. Susan Horwitz of its unique activity as a promoter of tubulin polymerization (Figure 6.1).3 Although clinically used drugs such as vinblastine (Velban) and vincristine (Oncovin) were known to act as inhibitors of tubulin polymerization, taxol was the first compound shown to act in the opposite direction. This discovery demonstrated that taxol was not “just another cytotoxic agent” and awakened a renewed interest in the compound — not least in the biological community — which turned out to be an important factor in keeping interest in taxol alive when it encountered problems during its initial clinical trials. C. CLINICAL DEVELOPMENT Taxol entered phase 1 clinical trials in 1984, but unfortunately it gave some allergic reactions, most probably related to its formulation as an emulsion with Cremophor EL®, a polyethoxylated castor oil. These reactions were responsible for at least one death.4 These problems would have spelled the demise of most drug candidates, but taxol’s unique mechanism of action was enough to encourage the clinicians to persevere, and the problems were overcome by lengthening the infusion period and premedicating patients. Ironically, these changes may have contributed to taxol’s eventual success, as there is evidence that the cytotoxicity and in vivo activity of taxol are increased on prolonged exposure to the drug.5 In any event, phase II trials, using the lengthened infusion period, were initiated in 1985, and these proved to be very successful. The first definitive clinical results were obtained in 1989 against drug-refractory ovarian cancer,6 and 2 yr later, excellent clinical results were reported against breast cancer.7 The drug was approved by the U.S. Food and Drug Administration for the treatment of refractory ovarian cancer in 1992 and for treatment of refractory or anthracycline-resistant breast cancer in 1994. The discovery and preclinical work as well as the initial clinical studies on taxol were sponsored by the NCI. Once it became clear that the compound had real therapeutic value, but that its further development would require more resources than NCI could allocate, its development was offered to the pharmaceutical industry under a Cooperative Research and Development Agreement Copyright © 2005 CRC Press, LLC HO O OH HO H O OH AcO OCOPh II.D.1 SCHEME 6.2 (CRADA). An agreement was signed with BMS in 1989, and because taxol was never patented, the CRADA gave BMS a 7-yr period of exclusivity in return for its development investment. D. THE TAXOL SUPPLY CRISIS The therapeutic efficacy of taxol became generally known by 1990, but unfortunately the supply of the drug from its natural source of T. brevifolia bark was not immediately adequate to the task of supplying a dramatically expanded demand. The situation was further complicated by the fact that T. brevifolia was primarily obtained from the old-growth forests of the Pacific Northwest of the United States, which forests were the natural habitat of the endangered spotted owl. The prospect of large-scale harvesting of T. brevifolia for taxol production thus raised serious environ- mental concerns.8 The initial approach to solving this vexing problem was to mount a large-scale collection and extraction program for T. brevifolia bark, which was successfully carried out by Hauser Chemical Research of Boulder, Colorado, under contract from BMS. This costly and labor-intensive approach was then replaced by a semisynthetic process from 10-deacetylbaccatin III (II.D.1), which Potier and his coworkers had found to be abundant in the European yew, Taxus baccata.9,10 Several groups developed methods to convert 10-deacetylbaccatin III to taxol, including those of Potier10 and Holton;11 the latter group’s method was selected by BMS for their commercial preparation of taxol. E. TAXOTERE This historical section would not be complete without a brief account of the discovery of docetaxel (Taxotere), the only other taxoid drug currently in clinical use. The Potier group at the Centre National de la Recherche Scientifique in Paris became interested in taxol in the early 1980s and carried out a series of isolation and semisynthetic studies. As noted above, an important initial finding was that the taxol precursor 10-deacetylbaccatin III (II.D.1) could be obtained in good yield from the needles of T. baccata — the English (or European) yew.9,10 Building on the availability of this compound, the group then developed various approaches to the semisynthesis of taxol from 10- deacetylbaccatin III. One of the first syntheses involved hydroxyamination of a cinnamoyl substituted baccatin III derivative to give a mixture of stereo- and regioisomeric hydroxyamines.12 Because the BOC group was being used as an amine protective group, one of these products was a protected version of compound II.E.1, and so this intermediate was converted to II.E.1 and tested for biological activity. It turned out to have excellent activity — better than taxol in some assays — and it was HO O OH O Me CO NH O 3 Ph O H O AcO OH OHOCOPh II.E.1 SCHEME 6.3 Copyright © 2005 CRC Press, LLC thus developed as a parallel drug to taxol. Taxotere, as the compound was named, entered phase I clinical trials in 199013 and was approved for treatment of advanced breast cancer in 1996 and for non–small cell lung cancer in 1999; its generic name is docetaxel.14 III. BIOSYNTHESIS AND BIOPRODUCTION OF TAXOL Interest in the biosynthesis of taxol has been driven in part by a need to develop a renewable and environmentally friendly source of this important compound. Significant work has been carried out both on the scientific study of the biosynthetic pathways to taxol and on the important practical application of plant tissue culture methods to the commercial production of the compound. This latter area of work has progressed significantly since a recent review,15 and taxol is now produced on a commercial scale by plant tissue culture by BMS at its plant in Ireland.16 A. TAXOL BIOSYNTHESIS 1. The Diterpenoid Ring System Taxol is a diterpenoid, and until a few years ago all diterpenoids were thought to arise from geranylgeranyl diphosphate, which was biosynthesized from mevalonic acid. In recent years, how- ever, a nonmevalonoid pathway to terpenoids has been uncovered,17 and it has been shown that at least some taxane diterpenoids are biosynthesized by this pathway. Thus, a feeding experiment with [U-13C6] glucose showed the incorporation of an intact 3-carbon precursor, eliminating the acetate-derived mevalonic acid as a precursor.18 The first committed step in the biosynthesis of the diterpenoid ring system of taxol is the cyclization of geranylgeranyl diphosphate (III.1) to taxa-4(5),11(12)-diene III.5.19 This step was thought initially to be the slow step, based on the low levels of III.5 found in T. brevifolia bark and the fact that the cyclization step is slow relative to subsequent oxygenations. Based on the presumed rate-determining nature of the cyclization of III.1 to III.5, the enzyme responsible for the cyclization, taxadiene synthase, was investigated in detail. The enzyme was purified from T. brevifolia bark and characterized as a 79-kDa protein.20 Genetic studies led to the isolation and expression of a cDNA fragment, which was expressed in Escherichia coli to produce the same protein.21 A study of the activity of taxadiene synthase in T. canadensis cell cultures showed that it was greater than that needed for taxol production in vivo, indicating that the true rate-limiting step of taxol biosynthesis occurs further down the pathway.22 The mechanism of the cyclization of III.1 to III.5 has been shown to involve an intramolecular

hydride ion transfer (presumably via an enzyme-bound intermediate) from the putative verticillyl ion III.2 to the cation III.3, which then undergoes intramolecular cyclization to the tricyclic cation III.4 and finally deprotonation to III.5.23 Because the taxane ring system is oxygenated at eight positions in taxol, the conversion of III.5 to taxol requires a series of oxidation reactions. A survey of the oxygenation patterns of known taxoids24 indicated the order of the oxygenation to be C5 and C10, then C2, C9, and C13, then C7 and C1, followed finally by epoxidation of the C4–C20 double bond and ring expansion to the oxetane ring. The first oxygenation step was shown to be formation of the C5 alcohol III.6;25 the low levels of III.6 found in T. brevifolia bark indicate that the hydroxylation of III.5 to III.6 may be the slow step of taxol biosynthesis. Conversion of III.6 to the acetate III.7 was demonstrated with a soluble enzyme preparation from T. canadensis, which is most probably the next step in the biosynthesis.26 It has proved to be difficult to elucidate the order of the hydroxylation reactions downstream of III.7, but a probable sequence is hydroxylation to the 10β-ol III.8 or the 13β-ol III.9,27 and thence to the tetrol III.10; the intermediacy of 2α,10β-diol derivatives has also been proposed.28 The conversion of taxusin (III.10, R = Ac) to its 7β-hydroxy derivative III.11 has also recently Copyright © 2005 CRC Press, LLC H H + + + H OPP H H H III.1 III.2 III.3 III.4 HO RO OR OR H H III.8 OR OR RO H H H H H H III.10 R = H or Ac OAc III.6 R = H III.5 HO H III.7 R = Ac H III.9 NH2 AcO O OH RO OROH AcO O OH O COOH C H NH O 6 5 III.13 OR O H O HO H O C H 6HRO 5 HO H R OBz OAc HO OBz OAc III.11 R = H or Ac III.12 III.14 R = H III.15R=OH SCHEME 6.4 been demonstrated.29 Substrate specificity studies with a set of available P450 hydroxylases indicate that there may well be more than one biosynthetic pathway to taxol in the yew, with the various pathways most probably converging on baccatin III (III.12). 30 Recently, cDNAs encoding two new P450 taxane hydroxylases and candidate genes for most of the steps of the taxol biosynthesis pathway have been identified. 31 2. The Side Chain The characteristic N-benzoylphenyl isoserine side chain of taxol has been shown to arise from phenylalanine, which was efficiently incorporated into taxol.32,33 The intact side chain was not incorporated into taxol, but the N-debenzoyl side chain was incorporated — although not as efficiently as β-phenylalanine.34 It is thus probable that hydroxylation of the side chain occurs after attachment to baccatin III, and that the major pathway is thus from α-phenylalanine to β-pheny- lalanine III.13. β-phenylalanine probably couples to baccatin III to give the 2-deoxytaxol III.14, and this finally undergoes hydroxylation to taxol (III.15). The overall pathway shown in Scheme 6.1 may be regarded as a plausible pathway to taxol, but the detailed steps, and especially the order of the various oxidation steps, have not yet been completely established. Steps that have been demonstrated are shown with solid arrows, and those that remain undefined are shown with dotted arrows. B. TAXOL BIOPRODUCTION The original source of taxol was the bark of the western yew, T. brevifolia, but this source is a limited and nonrenewable one, as the removal of the bark destroys the tree. In addition to this, T. brevifolia is concentrated primarily in the old-growth forests of the Pacific Northwest of the United Copyright © 2005 CRC Press, LLC States, and these forests are also home to the endangered spotted owl. The initial large-scale isolations of taxol for the licensee, BMS, were carried out by Hauser Chemical Research, Inc., and met with considerable controversy.9 In spite of this, large quantities of the drug were produced in this way until 1994, when BMS ended its contract with Hauser. The second phase of large-scale production began in 1993, with the adoption of a semisynthetic process for the synthesis of taxol from 10-deacetyl baccatin III (10-DAB), a compound that is available in yew needles in relatively large amounts.9,10 The conversion of 10-DAB to taxol involved a coupling reaction with a synthetic β-lactam intermediate; the details of this synthesis will be described later. Because 10-DAB is available from yew needles, which could in principle be harvested in a renewable way, this semisynthetic approach ensured a stable supply of taxol. The third phase of taxol’s bioproduction is that of plant tissue culture. An enormous effort was expended on the development of viable methods of taxol production by plant tissue culture methods, and this subject has been reviewed elsewhere.16,35,36 Production of taxol is enhanced by elicitation with methyl jasmonate and other elicitors, and yields of up to 115 mg/L in 2-week cell cultures of Taxus media grown in flasks have been reported.37 These yields are close to those needed for commercial production, and undoubtedly unpublished proprietary improvements have been made to facilitate large-scale production. The commercial production of taxol by cell culture methods has been reported in 2003 by the Korean company Samyang Genex,38 who name their product Genexol®. BMS also announced in 2003 that it is producing taxol by plant cell culture methods. The details of this process have not been released, but it is known that production is based on cultures of T. chinensis in Germany, and that final purification is carried out at the company’s plant in Swords, Ireland, based on a method developed in collaboration with Phyton, Inc.16 The method is reported to be much more environmentally friendly than the previous semisynthetic method, requiring only five organic solvents instead of the 13 solvents needed for the earlier method. The production of taxol by the endophytic fungus Taxomyces andreanae has also been reported,39 but the yield was extremely low (approximately 50 ng/L). More recently, the fungus Periconia sp. was found to produce taxol at the 800 ng/L level when stimulated with benzoic acid.40 However, at this point the maximum yields of taxol by the fungal route lag far behind those of the plant tissue culture route, and it has been suggested that the best prospects for microbial taxol production may require the discovery of endophytes that make large quantities of a precursor taxane that could be converted chemically to taxol.41 In summary, the commercial production of taxol has now moved to plant tissue culture methods, but semisynthesis from 10-DAB or other precursors will continue to be an important option for the commercial synthesis of the clinical candidates with modified structures described in section X. IV. TAXOL’S MECHANISM OF ACTION The discovery that taxol was a tubulin polymerization promoter3 was key to maintaining interest in it during its lengthy development, as at the time this was an unprecedented mechanism of action for an anticancer drug. Since that time, other natural products have been shown to have the same (or a very similar) mechanism, and at least two of these products, the epothilones and discoder- molide, are in clinical trials as anticancer agents. Each of these compounds is described in a separate chapter in this book and will thus not be discussed here. The primary mechanism of action of taxol has been investigated extensively. It promotes the polymerization of tubulin heterodimers to microtubules,42 binding with a stoichiometry of approx- imately 1 M taxol to 1 M tubulin dimmer. At high concentrations taxol both stabilizes microtubules and increases the total polymer mass,42 but these concentrations are higher than those needed to inhibit microtubule functions.43 At clinically relevant concentrations, taxol suppresses dynamic changes in microtubules, leading to mitotic arrest.44 Taxol interferes with the formation of the mitotic spindle, which causes the chromosomes not to segregate.45 Copyright © 2005 CRC Press, LLC Taxol has been shown to have many biological effects in addition to its ability to stabilize microtubules. Probably its most important activity after its microtubule assembly activity is the inactivation of the antiapoptotic protein Bcl-2. It does this by inducing phosphorylation,46 which leads to inactivation.47 The phosphorylation of Bcl-2 may occur through activation of Raf-1 kinase, and it has been proposed that Raf-1 is activated following drug-induced disruptions of microtu- bules.48 Taxol also binds directly to Bcl-2.49 In spite of taxol’s effect on Bcl-2 and other effects such as induction of the production of cytokines,49 it has been proposed that all the significant effects of taxol are directly traceable to its microtubule-binding activity; as a recent critical review states, “Unless convincingly proven oth- erwise, all relevant paclitaxel effects should be assumed to result from its microtubule-binding activity.”50 The actual effects resulting from microtubule binding are, however, rather complex. As another recent review states, “Depending on the phase of the cell cycle, taxanes can affect spindle formations, chromosome segregation, or completions of mitosis, thus activating the mitotic or the DNA-damage checkpoints and blocking cell-cycle progression. This complex scenario, with dif- ferent cell cycle responses related to the specific microtubule function affected in each phase, is reflected in the variety of pathways described to result in apoptosis upon taxane treatment. ”51 Because the most important biological effects of taxol can be attributed to its microtubule stabilization, the nature of its binding to microtubules becomes of great importance; this subject will be discussed in section VII of this chapter. V. MEDICINAL CHEMISTRY OF TAXOL The chemistry and medicinal chemistry of taxol have been extensively investigated, and it is not possible to discuss every aspect of this subject in the space allotted to this chapter. The emphasis of this section will thus be on work that has led to important new understandings or to compounds with improved activity. Readers interested in broader aspects of the subject are referred to any of several recent reviews.52–57 A. RING A MODIFICATIONS The C11(12) double bond in the A-ring of taxol is unreactive to hydrogenation; exhaustive hydro- genation of baccatin III gives a hexahydroproduct with a cyclohexylcarbonyl group replacing the C2 benzoyl group and an intact C11(12) double bond.58 It can, however, be epoxidized, and compound V.A.1 was prepared by epoxidation of 10-deacetoxytaxol. V.A.1 is more active than taxol in a tubulin-assembly assay but is less cytotoxic to B16 melanoma cells.59 A similar result was observed for the derivatives V.A.2 and V.A.3.60 The enol V.A.4 is surprisingly stable and can be converted to the analog V.A.5, which is slightly more cytotoxic than taxol.61 The C18 analogs V.A.6 (X = Me, N3, OAC, CN, or Br) could be prepared by allylic homination of 7,13-di(triethylsilyl) baccatin III followed by nucleophilic displacement and side chain attach- ment, but all were less cytotoxic than taxol.62 A-nortaxol analogs such as V.A.7 were prepared by treatment of taxol with Lewis acids;63 compounds of this type are much less cytotoxic than taxol, O OH O OH AcO O 1 OH O O R R2 O O Ph NH O Ph NH O Ph NH O Ph O H O Ph O H O Ph O H O OH AcO OH AcO OH AcO OH OCOPh OH OCOPh OH OCOPh V.A.1 V.A.2 R1 = R2 = bond X V.A.4 X= H V.A.3RR1=H,2 = F V.A.5 X = side chain SCHEME 6.5 Copyright © 2005 CRC Press, LLC O O O AcO X AcO OH AcO Ph NH O O O Ph NH O O OH OAc O NH O Ph O Ph O OH Ph O H O OH O OH OH AcO PhOCO PhCO O O OAc PhCO O OAc V.A.6 V.A.7 V.A.8 SCHEME 6.6 HO O OH AcO O OH O OH O O O NH O R NH O O HO H O O H O Ph O H O OH AcO OH O AcO AcO OH OCOPh O OCOPh OH OCOPh V.A.9 O V.A.10 V.A.11 R = Ph V.A.12 R = ButO SCHEME 6.7 although they do retain some tubulin-assembly activity. Similarly, compound V.A.8 with contracted A and C rings is noncytotoxic, but still shows tubulin-assembly promotion activity. The hydroxylated baccatin III derivative 14β-hydroxy-10-deacetylbaccatin III (V.A.9) was isolated from Taxus wallichiana leaves64 and has been used as the precursor for a series of 14β- hydroxytaxol analogs.65 The best of these turned out to be V.A.10, which is in phase II

clinical trials under the name ortataxel.56 Other compounds prepared from V.A.9 include the A-seco analogs V.A.10 and V.A.11, which were as active as taxol in the resistant cell line MCF7-R but were from 20 to 40 times less cytotoxic against several normal cell lines.66 B. RING B MODIFICATIONS Taxol can be selectively deacetylated at C10 by treatment with hydrazine67 or sodium bicarbonate and hydrogen peroxide,68 and it can be deoxygenated with samarium diiodide to give 10-deacetox- yltaxol V.B.1. Similar treatment of docetaxel gives the docetaxel analog V.B.2.69,70 Both 10- deacetoxyl71 and its docetaxel analog71 had similar cytotoxicity to taxol. Acylation of 10-deacetyl- taxol has yielded a number of 10-acyl analogs with improved bioactivities,72 although the best compounds (V.B.3 and V.B.4) also had modified side chains; these compounds were two orders of magnitude more active than taxol. The C9 position is occupied by a carbonyl group in taxol, but (9R)-13-acetyl-9-dihydrobaccatin III was isolated from T. canadensis and converted to 9-dihydrotaxol V.B.5.73 Various analogs of V.B.5 were also prepared.74,75 Deoxygenation of (9R)-13-acetyl-dihydrobaccatin III via the Barton procedure, followed by side chain attachment, gave the 9-deoxytaxol derivative V.B.6.76 O OH RO O OH AcO R OH O O O R NH O ButO NH O R NH O Ph O H O O H O Ph O H O OH HO AcO HO AcO OCOPh OH HO AcO OCOPh OH OCOPh V.B.1 R = Ph V.B.3 R = C2H5CO V.B.5 R = OH V.B.2 R = ButO V.B.4 R = cyclopropylcarbonyl V.B.6 R = H SCHEME 6.8 Copyright © 2005 CRC Press, LLC AcO O OH AcO O OH AcO O OH O O O Ph NH O Ph NH O Ph NH O Ph O H O Ph O H O Ph O H O OH HO R AcO OH HO AcO NHCOPh OH HO O AcO V.B.10 R = N V.B.7 R = H V.B.9 3 V.B.8 R = OCOPh V.B.11 R = OMe O R SCHEME 6.9 Modifications at the C2 position have yielded interesting results. The C2 benzoate group or a similar group is necessary for activity, and both 2-debenzoyloxy taxol77 (V.B.7) and 1-benzoyl- 2-debenzoyloxytaxol are both inactive.78 2-epi-Taxol (V.B.8) is also inactive,79 as is 1-benzoyl-2- debenzoyloxytaxol. On the other hand, 2-debenzoyloxy-2α-benzoylamidotaxol (V.B.9) retains cytotoxicity against three cell lines, with diminished activity as compared to taxol in two cell lines but with slightly increased activity in the third.80 Taxol analogs with substituted benzoyl groups at C2 can, however, be more active than taxol, although there is an interesting substituent effect on activity, in that para-substituted benzoate analogs are uniformly less active than taxol, whereas ortho- and meta-substituted analogs can be more active than taxol. Two of the best analogs are the m-azidobenzoyl derivative V.B.10 and the m-methoxybenzoyl derivative V. B.11.81,82 Ana- logs with 2-heteroaryl groups were also investigated, and thiophene carbonyl and furan carbonyl groups gave comparable activity to taxol.82–84 Most nonaromatic C2 esters at C2 gave analogs with lower activity, but the 3,3-dimethylacrylic ester showed better cytotoxicity against several cell lines.85 Direct deoxygenation of taxol at the C1 position has not proved possible, but a small series of 1-deoxytaxol analogs was prepared by modifications of baccatin VI.86 The analog V.B.12 was approximately half as active as taxol in a tubulin-assembly assay. C. RING C MODIFICATIONS The C7 hydroxy group is the most readily modified group in the diterpenoid ring system of taxol, and extensive modifications of it have been made. As the hydroxyl group of a β-hydroxyketone it is readily epimerized, and it can also be readily and selectively derivatized if the C2 hydroxyl group on the side chain is suitably protected. Some of the various acyl derivatives that have been prepared have had improved properties compared with taxol, and the DHA derivative V.C.187 and 7-hex- anoyltaxol V.C.288 are both currently in phase II clinical trial. Some simple ether derivatives also have had good activities; thus, thioethers V.C.3 and V.C.4 both had improved cytotoxicity and tubulin-assembly activity compared with taxol, and V.C.3 is currently in phase II clinical trials.89–91 Oxidation of the C7 hydroxyl group leads to the corresponding ketone, which is unstable in base and undergoes ring-opening to the αβ-unsaturated ketone V.C.5; this compound is devoid of activity.92 Deoxygenation at C7 has been achieved by several groups, and both 7-deoxytaxol V.C.693,94 and 10-acetyl-7-deoxydocetaxel V.C.795 have been prepared; these compounds are slightly less active than taxol in the tubulin assembly assay but are comparably cytotoxic to HCT 116 cells. HO O OH O ButO NH O Ph O H O H AcO OH OCOPh V.B.12 SCHEME 6.10 Copyright © 2005 CRC Press, LLC AcO O OR AcO O O R2O O O O O Ph NH O Ph NH O R1 NH O Ph O H O Ph O H Ph O H O H OAc OH HO AcO OH HO O HO AcO OCOPh OCOPh OH OCOPh V.C.1 R = COCH2(CH2CH=CH)6CH2CH3 V.C.5 V.C.6 R1 = C 6H , 2 5 R = CHCO V.C.2 R = CO(CH2)5CH3 V.C.7 R1 = ButO, R2 = H3 V.C.3 R = OCH2SCH3 V.C.4 R = SCH2OCH3 SCHEME 6.11 AcO O AcO O OH AcO O OH O O OH O OH Ph NH O Ph NH O Ph NH O Ph O H O Ph O H O Ph O H O OH HO AcO OCOPh OH HO AcO OCOPh OH HO AcO OCOPh V.C.8 V.C.9 V.C.10 SCHEME 6.12 Dehydration of taxol was achieved by treatment of the 7-triflate derivative with a nonnucleophilic base to give 6,7-dehydrotaxol V.C.8,96,97 which was comparable to taxol in its tubulin assembly and cytotoxic activity. Compound V.C.8 could be converted to the corresponding diol V.C.9 by osmy- lation, and this could be epimerized (albeit in low yield) to V.C.10, the major human metabolite of taxol.98 An improved synthesis of compound V.C.10 was reported by workers at BMS.99 Another interesting product was obtained by reaction of V.C.9 with lead tetraacetate to give V.C.11, but this compound was much less active than taxol.100 Two modifications of the C-ring of taxol have, however, led to compounds that have advanced to clinical trial or clinical candidacy. The first of these is the formation of a cyclopropane ring between C7 and C18. The cyclopropataxol V.C.12 and its docetaxel analog V.C.13 were discovered independently by workers at BMS101 and Rhone-Poulenc,102 and the analog V.C.14 is in phase II clinical trials as RPR-109881A. The second series of compounds is a surprising one, as it involves cleavage of ring C to give products that no longer maintain the rigid inverted-cup geometry of taxol. The compounds were discovered serendipitously by Appendino,103 but they showed good activity in the NCl 60-cell line panel and limited toxicity, and the C-seco analog V.C.15 has been selected as an antiangiogenic candidate for extended treatments.104 Various acyl derivatives of V.C.15 have been prepared, and these have similar activity to the parent compound, so in vivo oxidation and re-aldolization to the corresponding taxane derivative is excluded as the reason for the observed activity.105 Taxol analogs with modifications at C4 have also been prepared. A C4 acyl group is important for taxol’s activity, as removal of the C4 acetate leads to an inactive product.106 Reacylation of C4 AcO O OH R2O O O O Ph NH O R1 NH O O Ph O H OAc Ph O H O OH HO O HO AcO OCOPh H OCOPh V.C.11 V.C.12 R11 = C6Ht 5, R22 = CH3CO V.C.13 R1 = ButO, R2 = H V.C.14 R = BuO, R = CH3CO SCHEME 6.13 Copyright © 2005 CRC Press, LLC O OH OH AcO O OH O O ButO NH O Ph NH O O H O Ph O H O HO AcO OH OCOPh OH HO OCOOCH3 OCOPh V.C.15 V.C.16 SCHEME 6.14 AcO O OH R2O O OH O O Ph NH O R1 NH O OH Ph O H Ph O H X HO HO OAc R3O OH OCOPh OH HO OCOPh V.D.1 V.D.2 R1 = ButO, R2 = H, R3 = COCH3, X = NH V.D.3 R1 = Ph, R2 = OCOCH3, R 3 = COOCH3, X = S SCHEME 6.15 with various acyl groups gave some products with improved activity, and the C4 carbonate V.C.16 is in Phase II clinical trials.107,108 Various analogs with C2 and C4 acyl modifications have also been made, and some of these compounds showed enhanced activity against resistant cell lines.109 D. RING D MODIFICATIONS The oxetane ring is an unusual feature of the taxol structure, and it plays a significant role in taxol’s unique activity. Thus, opening of the oxetane ring to a compound such as V.C.5 destroys taxol’s activity, as does ring opening by electrophilic reagents to give compound V.D.1.63 Taxol analogs in which the oxetane oxygen atom is replaced with nitrogen (V.D.2)110 or sulfur (V.D.3)111 are much less active than taxol. The importance of the oxetane ring cannot be because of its chemical reactivity, as taxol binds noncovalently to tubulin. It is thus probable that it serves as a conformational lock, holding the diterpenoid ring system in its characteristic inverted-cup conformation, and also (in conjunction with the C4 acetate) as a hydrogen bond acceptor. These conclusions were put on an quantitative basis by Snyder and his colleagues,112 who predicted that other functional groups would be capable of assisting the binding of taxol to tubulin as effectively as the oxetane ring. This prediction was borne out by the synthesis of the cyclopropane derivative V.D.4, which is almost as active as taxol in a tubulin assembly assay.113 Compound V.D.4 contains both the necessary C4 acetate and rigid- ifying cyclopropane ring, and both features appear to be necessary for activity. Compound V.D.5, which contains only the C4 acetate function, is much less active than taxol.114 Similarly, compound V.D.6, with both C4 and C20 acetate functions, is also inactive as a tubulin assembly agent.115 AcO O O HO O OH AcO O OH O O O ButO NH O ButO NH O Ph NH O Ph O H Ph O H Ac Ph O H HO OAc HO AcO OAc OH OH HO O OCOPh OH OCOPh OCOPh V.D.4 V.D.5 V.D.6 SCHEME 6.16 Copyright © 2005 CRC Press, LLC AcO O OH HO O OH HO O OH O O NH O O Ph NH HO H O O H O O H O HO OH OH AcO Ph O AcO OH AcO OCOPh OH O OCOPh O V.E.1 V.E.2 V.E.3 R = C6H5 N3 V.EE.4 R = ButO SCHEME 6.17 E. SIDE CHAIN ANALOGS The side chain of taxol is in many ways the most readily modified part of the molecule, as it can be prepared synthetically and linked to baccatin III by one of the various methods described in section VI.A. It has been found that the 3′-phenyl and N-benzoyl groups of taxol are not specifically required for activity, and several of the taxol analogs in clinical development or in clinical trials have modified side chains.116 Space does not permit a detailed discussion of all the side chain variations that have been investigated; more complete details can be found in the reviews previously mentioned.52–56 The side chain itself is necessary for the full activity of taxol, and all the compounds in clinical trial have a substituted isoserine side chain of some nature. However, suitably modified baccatin III derivatives can retain significant activity, as is the case with 2-(m-azidobenzoyl) baccatin III (V.E.1).117 Not only is the side chain necessary for full activity, its regio- and stereochemistry are also indispensable. Analogs with simplified side chains, such as N-benzoylisoserine or phenyllactic acid,118 are significantly less active, and analogs with different stereochemistries than the 2R, 3S stereochemistry of taxol are also less active than taxol.119 Some analogs with conformationally restricted side chains have been prepared, and compound V.E.2 is only moderately less active than taxol.120 Extension of the side chain by one carbon, as in V.E.3 and V.E.4, caused a 30-fold loss of tubulin assembly activity,121 whereas transposition of the hydroxyl and amino substituents also caused a reduction in activity by a factor of about ten.119 Modifications of the 3-phenyl and N-benzoyl substituents have been more fruitful. Replacement of the 3′-phenyl group with various substituted phenyl groups is largely unproductive, but replace- ment with moderately sized alkyl groups such as isobutyl or isobutenyl gives compounds with improved

activity,122 and as noted earlier, the analog V.A.10 with this substitution is in clinical trials. Replacement of the 3′-phenyl with a 3′-(2-furyl) group also gives improved activity,72 and the compound MAC-321 (also designated TL-00139, V.E.5) is in clinical trials.123 The 3-(fluoro- pyridyl) group also imparts improved activity, and the analog V.E.6 is in clinical trials,124 and a final example is the 3′-(t-butyl) analog V.E.7, which is as active in mice when given orally as taxol is when given intravenously.125 O N HO O O O O AcO O OH O O O O NH O O NH O O NH O O H O N O H O O H O O OH OH OAc OH OH AcO OH OCOOCH OCOPh F OCOPh OH 3 OCOPh V.E.5 V.E.6 V.E.7 SCHEME 6.18 Copyright © 2005 CRC Press, LLC AcO O OH AcO O OH HO O OH O O O O NH O O NH O O NH O Ph O H O O O H O Ph O H O OH OH AcO OH O AcO F F OH AcO OCOPh O OCOPh OCOPh V.E.8 O V.E.9 V.E.10 SCHEME 6.19 Various modifications of the 3′-N substituent have been studied. An acyl substituent at this position is required for activity, and the t-butoxycarbonyl group of docetaxel (Taxotere, II.E.1) has been proven an effective alternative acyl group to the benzoyl group of taxol. All the taxol analog clinical candidates surveyed in a recent review116 have a benzoyl group, a t-butoxycarbonyl group, or in one case, a substituted benzoyl group as the 3-N substituent. The 2′-hydroxyl group was early on shown to be necessary for activity,126 and successful modifications (other than acylation to give a prodrug, discussed in the next sections) have been rare. A few cases of important activity have been discovered, however. Several 2′-methyl taxol derivatives such as V.E.8 have been prepared and have been found to have improved cytotoxicity as compared with taxol.127–129 A recent paper describes the synthesis of the analog V.E.9, which is more active than taxol in both normal A2780 cells and taxol-resistant A2780 cells.130 Replacement of the 2-hydroxyl group with a thiol group gave an inactive compound,131 but the 2,2-difluoro analog V.E.10 is somewhat more active than taxol toward P388 cells.132 F. PRODRUGS OF TAXOL The low water-solubility of taxol was a major problem in its early development, so the preparation of water-soluble prodrugs was an important part of the early work on the drug. The development of more active and more water-soluble analogs of taxol described in the preceding sections and summarized in section IX below has rendered much of the prodrug work obsolete. At present the only prodrug that is included in a recent survey of taxanes in development116 is T-3782 (V.F.1; compound 4a in Yamaguchi et al.133). This section is thus abbreviated and will only mention a few of the variations that have been prepared. A more detailed treatment can be found in the reviews previously mentioned.52,134 Initial studies focused on simple ester derivatives at the 2-position, as these are readily hydro- lyzed to taxol. Various succinate and glutarate derivatives were prepared,126,135 as wall as sulfonic acid salts136 and amino acid derivatives.126,137 The prodrug V.F.1 noted above is of this general type, but with modified side chain substituents as well as the amino acid linked through a glycolate spacer. Phosphate prodrugs have proved attractive because of phosphatases present in cells, and inge- nious use was made of this fact in the synthesis of prodrugs such as V.F.2; this compound is stable in water but releases taxol by internal lactonization after dephosphorylation by phosphatases.138 Regrettably, V.F.2 was found to bind to plasma proteins and was deemed to be unsuitable for use as a prodrug, although it did show good in vitro activity against the murine M109 tumor.138 A second ingenious solution to the water-solubility problem was developed by noting that the O-benzoyl to N-benzoyl migration of 2′-benzoyl-3′N-debenzoyltaxol (V.F.3) is slow at pH 4.0. This compound is much more water soluble than taxol at this pH and can thus be used as a prodrug, with conversion to taxol occurring relatively quickly (t∫ = 15 min) under physiological conditions.139 The same strategy has recently been applied to generate V.F.4, a prodrug of canadensol.140 G. TARGETED ANALOGS OF TAXOL Although the need for prodrugs of taxol has been alleviated by the development of more potent water-soluble analogs, there is still a need to develop analogs that can be delivered more selectively Copyright © 2005 CRC Press, LLC O OPO3Na2 HO O OH AcO O O AcO O OH O O O NH O Ph NH O NH2 O O H O Ph O H O Ph O H O O OH OAc O OH OH OAc O OCO h OCOPh O O OH OAc P OCOPh O NH R V.F.3 R = Ph 2 V.F.1 V.F.2 V.F.4 R = CHMe O NH 2 2 SCHEME 6.20 to the tumor cells. Some of the methods for doing this involve new pharmaceutical formulations and will be discussed in section IX. In other cases, targeting agents have been covalently linked to taxol, and these modifications are described here. Several targeting analogs make use of monoclonal antibodies (mAbs) to target taxol to a tumor site. In the antibody-directed enzyme prodrug therapy (ADEPT) approach,141 specific enzymes are delivered to tumor sites by mAbs, and these enzymes are then available to activate a “protected” taxol derivative selectively cleaved by this enzyme, resulting in release of taxol. The taxol analog V.G.1, for example, can be converted to taxol by hydrolysis of the glucuronide with a glucuronidase, decarboxylation, and internal lactam formation.142 A p-nitrophenol linker has also been used with similar results,143 and docetaxel has also been targeted by this approach.144 A second strategy makes use of the fact that the protease plasmin plays an important role in tumor invasion and metastasis and is thus present in tumors. Taxol prodrugs were thus prepared in which plasmin substrate peptides were linked through a spacer to the 2-position of taxol. One of the best such compounds is V.G.2: This compound is one of the least toxic taxol prodrugs reported, and yet it is converted by plasmin to taxol with a half life of 42 min.145 Another tumor-associated enzyme is neuraminidase, and the taxol-sialic acid hybrid V.G.3 was prepared as a neuraminidase-cleavable prodrug.146 Compound V.G.3 was somewhat less cytotoxic than taxol, but it was much more water-soluble. No information on its cleavage by neuraminidase was provided. A taxol-hyaluronic acid conjugate has been prepared in which the polymeric hyaluronic acid is linked to taxol through a linker at the 2-position.147 The conjugates showed selective toxicities toward breast, colon, and ovarian cancer cell lines that are known to overexpress hyaluronic acid receptors, but were nontoxic to a mouse fibroblast cell line. AcO O OH AcO O OH O O Ph NH O Ph NH O Ph O H O Ph O H O O OH OAc O OH OAc O-Na+ O OCOPh O OCOPh O HO O H N O HO N OH O V.G.1 V.G.2 O NH Cl- H H3N + N N H O O Cl-H + 3N SCHEME 6.21 Copyright © 2005 CRC Press, LLC OH HO COOH OH AcNH O O O O HO HN AcO O O O AcO O OH O O Ph NH O Ph NH O Ph O H O Ph O H O OH OH OAc O OH OAc OCOPh OCOPh V.G.3 O V.G.4 SCHEME 6.22 A different approach was taken in the synthesis of the docosahexaenoic acid conjugate V.G.4, based on the observation that tumors rapidly take up some fatty acids from arterial blood. This compound was evaluated in a phase I clinical trial and was found to be well tolerated by the patients. It also proved to be curative for mice with the M109 tumor, in contrast to taxol, which was not curative in this system.148,149 The C7 analog V.C.1 mentioned previously is also a targeted analog of this type. Folic acid is another compound that is taken up selectively by cancer cells, and taxol-folic acid conjugates have been prepared and evaluated.150 Compound V.G.5 was found to increase the life span of mice with the M109 tumor, but it was disappointingly less effective than taxol itself. It has been found that short oligomers of arginine exhibit superior membrane translocation activity, and a series of arginine-based molecular transporters of taxol has been prepared.151 The general structure of these compounds is V.G.6; they are much more water soluble than taxol and release free taxol with half-lives of minutes to hours, depending on the linker structure and the pH of the medium. The final and most direct strategy is that of linking taxol directly to a mAb. This approach has been adopted by more than one group, with promising results. Thus, conjugation of taxol to the anti- p75 mAb MC192 was achieved through a 2-glutaric acid linker, and the resulting conjugate had improved cytotoxicity and improved selectivity as compared to taxol.152 In another approach, several taxane–antibody immunoconjugates have been prepared by Ojima.153 As one example, the conjugate V.G.7 was prepared using an antibody that recognized the human epidermal growth factor (EGFR) expressed in human squamous cells. The conjugate was shown to have excellent activity in vivo against EGFR — expressing A431 tumor xenografts in severe combined immunodeficiency (SCID) mice.153 In summary, the targeting of taxol and other taxanes through the approaches described above offers a way to improve therapeutic effect and reduce toxicity, and it seems probable that some targeted analogs will be introduced into clinical trials within the next few years. H. SUMMARY OF TAXOL’S SAR A summary of the SARs of taxol describes in the preceding sections is provided in structural form in Figure 6.2. O CH3 O O O N O AcO O O N N OH O O H 3 H HN N N NH Ph NH O O H N N NH Ph O H O 2 OH OAc OH OCOPh V.G.5 SCHEME 6.23 Copyright © 2005 CRC Press, LLC AcO O OH O O Ph NH O N mAb H O H S Ph O O S O O OH O OH OAc O O OCOPh O NH O S O O H O H N CONH O OH OAc 2 Arg H 8 2 OCOPh 4-5 V.G.6 V.G.7 SCHEME 6.24 VI. SYNTHETIC STUDIES A. SEMISYNTHETIC METHODS Much of the work described in the preceding sections has only been possible because synthetic methods are available for the attachment of the taxol side chain to baccatin III. In addition, until recently, taxol itself was manufactured on a large scale by semisynthesis from baccatin III, lending a large incentive for the discovery of improved or alternate methods for its synthesis. All syntheses of taxol and its analogs start with 10-DAB (II.D.1), which must be protected at C7 and C10 before acylation at the C13 position. If a 10-acyl compound such as taxol is the desired product, 10-DAB is normally protected as its 7-triethylsilyl ether and then selectively acylated at C10 before reaction with a suitable side chain precursor.154 Many different approaches have been used to couple the side chain to protected baccatin III, and only the most important will be described here. The most important method from a commercial point of view is that which is based on β-lactam chemistry, as this was the method used by BMS in their semisynthesis of taxol until it was replaced by the plant tissue culture production method. Acetyl or acetoxy group may be N-acyl group removed without required; Reduction significant loss May be esterified, some acyl improves of activity; some epimerized, or analogs have activity acyl analogs removed without improved slightly have improved significant loss of activity activity activity. Some AcO O OH derivatives have O improved activity C6H5 NH O Oxetane or cyclopropane ring required for activity; H O substitution of S for O C6H5 O reduces activity OH AcO OH O O Group may be changed Removal of acetate reduces activity to alkenyl or substituted Free 2'-hydroxyl group, C slightly. Replacement by other groups 6H5 phenyl. Some groups or a hydrolysable ester can increase activity give improved activity. thereof, required Acyloxy group essential; certain alkenyl and substituted aromatic groups give improved activity Contracted A-ring inactive Hydroxyl group

helpful but not essential 14β hydroxyl group or 14β.1 carbonate analogs retain activity FIGURE 6.2 The Structure-Activity Relationships of Taxol. Copyright © 2005 CRC Press, LLC OSiiPr Ph R3SiO Ph LiO N N Ph Me3Si O Ph OSiMe2But O O (C6H11)2NO2S VI.A.1 VI.A.2 VI.A.3 VI.A.4 SCHEME 6.25 The chemistry was developed independently by Holton11 and Ojima155 and is a simple and efficient route to taxol. The key nonracemic β-lactam VI.A.1 can be prepared by various methods, all of which involve a [2+2] cycloaddition reaction. Two different types of approach can be used, depending on whether the chiral auxiliary is associated with the enolate or the imine half of the cycloaddition reaction. Methods that use a chiral enolate include the use of a 2-phenylcyclohexanol enolate (VI.A.2 + VI.A.3)156 and of a chiral enolate of Oppolzer’s auxiliary (VI.A.4).157 A chiral oxazolidinone enolate has also been used, although this approach requires several steps to remove the chiral auxiliary when it is no longer needed.158,159 Chiral imines have also yielded diastereoselective 2+2 cycloadditions. The imine VI.A.5 gave 84% diastereomeric excess of the desired β-lactam, although several steps were required to remove the chiral auxiliary.160 The imine VI.A.6 gave a 50%–60% diastereomeric excess of β-lactam, but this had to be hydrolyzed to the open-chain amino acid to remove the chiral auxiliary by hydro- genolysis.161 A better diastereomeric excess was achieved with the imine VI.A.7.162 The β-lactam can also be prepared selectively from an α-bromoamide precursor; thus treatment of the intermediate VI.A.8 with NaH or TBAF gave β-lactam in good yield without significant loss of optical purity.163 An alternative and experimentally simpler approach is to prepare the acetate of racemic β-lactam VI.A.9 by simple 2+2 cycloaddition and then resolve it with a commercially available lipase.164 Ironically, all this effort to prepare enantiopure β-lactams may be unnecessary, as Ojima has shown that kinetic resolution can be obtained in coupling racemic β-lactams with baccatins.165 Coupling of the protected β-lactam, however prepared, with protected baccatin III is an efficient and high-yield reaction. Coupling is normally carried out in the presence of NaH or LiHDMS, and yields greater than 90% are routinely achieved. Thus, coupling of β-lactam VI.A.1 with the protected baccatin III derivative VI.A.10 gives the protected taxol derivative VI.A.11, which can be depro- tected to give taxol I.1. Ph Ph Ph Ac OSiPh2But O Ph O Br O N N Ph N O N Ph O Ph NHCH2Ph O COOMe O OAc O VI.A.5 VI.A.6 VI.A.7 VI.A.8 VI.A.9 SCHEME 6.26 AcO O OSiEt A O 3 cO OSiEt AcO O O R O 3 O H 3SiO Ph Ph NH O Ph NH O N Ph O HO H O Ph O H O Ph O H O O OH AcO OCOPh OSiR OH AcO OH AcO 3 OCOPh OH OCOPh VI.A.1 VI.A.10 VI.A.11 I.1 SCHEME 6.27 Copyright © 2005 CRC Press, LLC AcO O OCOOCH2CCl HO O OCOOCH2CCl3 cO O 3 A O O H O COOH O Ph NH O N O Ph O Me OCO HO H O H O Ph O H O 3C OH AcO N OH AcO OH OH AcO VI.A.12 OCOPh Me3COCO Ph OCOPh OCOPh VI.A.13 VI.A.14 I.1 SCHEME 6.28 Most of the other approaches to taxol from baccatin III also involve cyclic intermediates, as these minimize the steric constraints inherent in acylating the very hindered C13 hydroxyl group of baccatin III. Groups that have been used successfully include the oxazolidine group, such as VI.A.12, which has been coupled with the protected baccatin III VI.A.13; the coupled product VI.A.14 must then be deprotected by treatment with formic acid and benzoylated to give taxol. Oxazolines such as VI.A.15 can also be coupled in high yield; hydrolysis of the intermediate oxazoline VI.A.16 gives taxol I.1. As is also the case with the β-lactam approach, this route is atom economical in that the protecting group is part of the final product.166,167 Many other methods have been developed for the synthesis of taxol and related compounds from baccatin III. These include coupling of oxazolidine thioesters,168 coupling with cis-glycidic acids followed by appropriate manipulations,169 and coupling with a β-ketoester and subsequent functionalizations.170 B. TOTAL SYNTHESIS The structure of taxol, with its complex stereochemistry and unique ring structure, represents one of the most challenging natural product synthetic targets. In spite of this complexity, six independent total syntheses have been achieved, together with numerous partial syntheses and unique synthetic approaches. Because of space limitations, and also because this subject has been recently reviewed elsewhere,53,171 only a general summary of the synthetic routes will be presented here. Also, because baccatin III can be converted to taxol by any of the routes described in the preceding section, a synthesis of baccatin III constitutes a formal synthesis of taxol. The first two syntheses were published essentially simultaneously by Holton172,173 and Nico- laou.174 The Holton synthesis started with the natural product β-patchoulene, which eventually gave the A and B rings, which were subsequently elaborated into the final product. The strategy was thus a linear one, of the form AB→ABC→ABCD. β-Patchoulene was converted to the protected diol VI.B.1 and thence through a clever ring opening of its epoxide to the bicyclic AB ring system VI.B.2. This was then converted to the carbonate VI.B.3, and thence to the ABC ring system VI.B.4. Final elaboration of ring D and appropriate functional group manipulations gave baccatin III.173 This synthesis gave the unnatural enantiomer (+)-taxol if the commercially available β-patchoulene was used, but the natural (–)-taxol enantiomer could be prepared if (–)-borneol was used as starting material, although this required additional synthetic manipulations. The Nicolaou synthesis174 used the synthetic design A+C→A–C→ABC→ABCD. The pre- formed A (VI.B.5) and B (VI.B.6) rings were coupled by a Shapiro reaction to give the A–C AcO O OSiEt AcO O 3 OH O Ph COOH O Ph NH O N O Ph O H O Ph O H O N O OH AcO OH AcO Ph OCOPh OH OCOPh VI.A.15 VI.A.16 I.1 Ph SCHEME 6.29 Copyright © 2005 CRC Press, LLC Et3SiO Et3SiO Et3SiO OBOM O OSiEt OH 3 O O O ButMe2SiO ButMe2SiO ButMe2SiO H OTMS O O O O VI.B.1 VI.B.2 VI.B.3 O VI.B.4 SCHEME 6.30 ButMe2SiO O O Pri 3SiO OBn OBn HO OH OBn AcO O OSiEt3 OHC H O NNHSO H O 2Ar O H O H O O O O O O O O O OH O O O VI.B.5 VI.B.6 VI.B.7 VI.B.8 VI.B.9 SCHEME 6.31 intermediate VI.B.7. This was then converted into the ABC system (VI.B.8) by a McMurry coupling; this reaction proved to be the Achilles’ heel of the synthesis, with a yield of only 23%. Compound VI.B.8 was then elaborated to baccatin III. The Danishefsky synthesis175 is of the form CD + A→A–CD→ABCD and is the only synthesis in which the oxetane ring is formed at an early stage and carried through the entire synthetic sequence. This was possible because the C4 hydroxyl group was protected as its benzyl ether, thus avoiding complications from neighboring group participation by the C4 acetate. Coupling of the A-ring synthon VI.B.10 to the CD-synthon was achieved by lithiation of VI.B.10 and 1,2-addition to the aldehyde group of VI.B.11. Cyclization to the ABCD system VI.B.13 was brought about by a Heck reaction. Functional group manipulations converted VI.B.13 to VI.B.14 and the latter compound to baccatin III. The Wender synthesis176,177 is similar to the Holton synthesis in being a linear synthesis of the form A→AB→ABC→ABCD. The starting material was verbenone, which was converted by some clever chemistry into VI.B.15. Oxidative cleavage of this compound was carried out by a method related to the Holton conversion of VI.B.1 to VI.B.2 and gave VI.B.16. This compound was then converted to VI.B.17 through elaboration of the C3 substituent, and aldol condensation of this formed the C ring. The synthesis was completed through intermediate VI.B.18 and formation of the oxetane ring. The overall synthesis, at 37 steps from verbenone, is the shortest recorded synthesis of taxol to date. MeO OSiMe But MeO OMe OSiMe OSi e B O OSiEt MeO 2 O M ut OSiMe But 3 2 2 3 CN I H O H O H O H O O BnO HO BnO O O OBn HO OAc OCOC H O 6 5 VI.B.10 VI.B.11 VI.B.12 VI.B.13 VI.B.14 SCHEME 6.32 Copyright © 2005 CRC Press, LLC Taxol and Its Analogs 109 O O O OSiEt3 O AcO O OTroc O O OBOM OBOM HO TIPSO Pri O OSiMe 3SiO CH S Me But 2But Pri Si H O O 3 O HO 2O i 2 OCOC6H5 O VI.B.15 VI.B.16 VI.B.17 VI.B.18 SCHEME 6.33 OSiPri OSiPri 3 PhS OBn O OMOP 3 Me B O HO SPh O Li SPh CH(OBn) OHC O Me2ButSiO H 2 CH2(OBn)2 HO OH O O O C6H5 VI.B.19 VI.B.20 VI.B.21 VI.B.22 VI.B.23 SCHEME 6.34 The Kuwajima synthesis was reported in a preliminary communication in 1998, with the full report appearing in 2000.178 It is of the form A + C→A–C→ABC→ABCD, and uses a 1,2-addition of the organolithium VI.B.19 to the aldehyde group of VI.B.20 to form the key A–C bond in VI.B.21. The second ring closure to form the B ring in VI.B.22 was brought about by Lewis acid catalysis, with subsequent conversion to baccatin III occurring via the diketone VI.B.23. The Mukaiyama synthesis took the unusual pathway of forming the B ring first, and is of the form B→BC→ABC→ABCD.179 Ring B was formed by cyclization of the linear precursor VI.B.24 with Sml2, and ring C was added through conjugate addition of a lithium cuprate reagent to the αβ-unsaturated ketone of VI.B.25. Ring A was added by addition of an organolithium reagent to the ketone of VI.B.26, and ring D was formed by cyclization of the diol precursor VI.B.27. As noted earlier, all of these syntheses stand out as major synthetic achievements, but none of them is new knowledge of taxol’s SARs capable of providing an economical approach to the large- scale synthesis of taxol. Fortunately, the plant tissue culture methods described earlier have provided an alternate and environmentally friendly way of producing taxol, but the synthetic efforts have nevertheless been very useful as platforms for the development of new chemistry. VII. THE TAXOL–TUBULIN INTERACTION As noted in section IV, the primary mechanism of action of taxol is its ability to stabilize micro- tubules and thus to disturb the equilibrium between tubulin and microtubules that is so essential BnO O BnO O Br BnO OBn O AcO O O Me2ButSiO Me2ButSiO HO HO O OPMB OSiMe t Me t 2Bu 2Bu SiO H H Br PMBO O OH OBn OBn O O OH O VI.B.24 VI.B.25 VI.B.26 VI.B.27 SCHEME 6.35 Copyright © 2005 CRC Press, LLC for normal cellular function. The nature of interactions between taxol and tubulin has thus been a subject of intense interest, and many different tools have been used to study it. The location of the taxol binding site within the tubulin molecule was shown to be on β-tubulin by photoaffinity labeling studies with 3-N-debenzoyl-3-N-(p-azidobenzoyl) taxol ([3H]-VII.1), which photolabeled the N-terminal proton of β-tubulin.180 In contrast, 2-debenzoyl-2-(m-azidoben- zoyl) taxol (VII.2) labeled amino acids 217-231 of β-tubulin,181 and taxol with a photoaffinity label at C7 bound to Arg282 in β-tubulin.182 Although β-tubulin is the tubulin molecule that is labeled most extensively by photoaffinity- labeled taxol, some analogs do label α-tubulin as well as (or instead of) β-tubulin. Thus [3H]-VII.1 does bind to α-tubulin to a modest extent,183 and [3H]-VII.3 labeled amino acids 281–304 of α- tubulin.184 These results thus indicate that taxol most probably occupies a binding site located between α- and β-tubulin, but largely on β-tubulin. In ideal circumstances the binding site of taxol on tubulin would be elucidated by x-ray crystallography, but it has not been possible to obtain crystals of the polymeric complex. It has proved possible, however, to obtain electron diffraction data on the αβ-tubulin-taxol complex as zinc-induced sheets, and this has proved enormously important in understanding the taxol–tubulin interaction.185,186 In this structure, taxol occupies a hydrophobic cleft on β-tubulin, and the binding of taxol in this cleft converts it into a hydrophilic surface.187 This binding location is consistent with the photoaffinity results reported above, showing labeling of β-tubulin by taxol; the labeling of α-tubulin is difficult to explain. Although the electron crystallographic results show the location of

taxol on tubulin, the actual binding conformation of taxol cannot be discerned at the resolution obtained, and indirect methods thus must be used to make this determination. A knowledge of the binding conformation of taxol is important, in that it would help to explain how compounds as structurally diverse as the epothilones, discodermolide, and eleutherobin could all have the same mechanism of action, and it could also provide a guiding model for the synthesis of simplified compounds that retained tubulin-binding ability. Information on the binding conformations of taxol has been obtained by two studies using solid-state nuclear magnetic resonance (NMR) to probe the conformation of tubulin-bound taxol. In the first study, an F–F distance of 6.5 Å was determined for the compound VII.4,188 and in the second, F–13C distances of 9.8 and 10.3 Å were determined for the distances to the C3 amide carbonyl and the C3 methine carbons, respectively, of VII.5.189 Studies of the NMR spectra of taxol in solution indicated the presence of two major conformers — a nonpolar conformer with the C2 benzoyl group and the C3 phenyl side chain exhibiting hydrophobic collapse190–192 and a polar form in which the C2 benzoyl group and C3 benzamido side chain are in close proximity.1193–195 However, NMR deconvolution analysis in chloroform also revealed the presence of a third conformer, designated T-taxol, in which the C2 benzoyl group bisects the angle between the C3 phenyl and C3 benzamido side chains.196 The T-taxol conformation is consistent with the solid-state NMR studies previously referred to and also with the unexpected activity of 2- m-azidobaccatin III.197 It also docks well into the hydrophobic cleft in β-tubulin described earlier.187 AcO O OH N A O 3 cO AcO O OH O OH O O R2 x NH O N C y NH O R2 NH O Me x R1 Ph O H O Ph O H O C O H O OH AcO OH OH AcO OH O OH OH AcO O OCOPh R1 O O VII.1 R1 R3 = N3, R2 = R3 = H VII.3 VII.4 R1 = Me3CO, R2 = F, x = 12, y = 14 VII.2 R1 = R VII.5 R1 = Ph, R2 = H, x = 13, y = 15 F 2 = H, R3 = N3 SCHEME 6.36 Copyright © 2005 CRC Press, LLC AcO O OH AcO O OH HO O OH OH O Ph O O H O HO O H O O O H O H Ph N OH AcO OH AcO O OH AcO O O HN O OH O ButO N O O H O n n' O O X Y VII.7 X = Y = CH2 VII.9 VII.10 VII.8 X = CO, Y = O SCHEME 6.37 Tests of the various models have been made using the synthesis of conformationally constrained taxols. A test of the hydrophobically collapsed model was made by the synthesis of compounds VII.7 and VII.8, and both of these compounds were completely inactive in a tubulin assembly assay, indicating that taxol does not adopt a hydrophobically collapsed conformation in the bound state.198 A second series of compounds was based on the proposal of a common pharmacophore for taxol, the epothilones, discodermolide, and eleutherobin.199 The analog VII.9 was prepared based on this model, and it did show cytotoxic activity against the MDA435/LCC6-WT cell line, but it was over 100-fold less active than taxol. It also was active as a tubulin-assembly agent, with 37% of the activity of taxol. A third approach to an active macrocyclic taxol analog is exemplified by the compound series VII.10, where n and n′ range from 2 to 4.200 The best of these compounds was sevenfold less active than taxol as a tubulin-assembly agent but was over three orders of magnitude less cytotoxic. All of the bridged analogs described above are linked from the side chain to the C2 positions. A different series has been prepared on the basis of the T-taxol design concept, and this series has been spectacularly successful in providing bridged analogs that not only equal but surpass taxol in its activity.201 A key compound is the bridged taxol VII.11; this compound is about 20-fold more cytotoxic than taxol to A2780 cells and has a critical concentration for tubulin polymerizations over threefold lower than taxol’s. The compound maps well onto the T-taxol structure and fits the taxol-binding pocket on β-tubulin as well as taxol itself. This structure thus represents the best available model of the tubulin-bound conformation of taxol and presents an attractive target for the synthesis of simplified analogs that retain the active T-conformations of taxol. VIII. OTHER NATURAL PRODUCTS WITH A SIMILAR MECHANISM OF ACTION In recent years several natural products with a similar mechanism of action to taxol’s have been discovered, and some of these have been developed as clinical candidates or are in clinical trial. These include the epothilones A (VIII.1) and B (VIII.2),202,203 discodermolide VIII.3,204 and AcO O OH O HO O H O O HO O BzO N H O VII.11 SCHEME 6.38 Copyright © 2005 CRC Press, LLC OH O R HO O S O O AcO O N OH OH OH O MeO H O O OH OH NH O 2 O OH O N H N O VIII.1 R = H VIII.2 R = Me VIII.3 O VIII.4 SCHEME 6.39 eleutherobin VIII.4.205 These epothilones and discodermolide are discussed in more detail in separate chapters in this volume. Other compounds with similar mechanisms of action include rhazinilam,206 which inhibits the disassembly of microtubules by a different mechanism than taxol, laulimalide and isolaulimalide,207 WS9885B,208 polyisoprenylated benzophenones related to guttif- erone E,209 jatrophane esters,210 dictyostatin,211 and even some steroid derivatives212 and dicouma- rol.213 Several reviews covering this area have appeared.214–216 The tubulin-bound conformations of these agents has proved a fruitful field for modeling and computational approaches. Several authors have advanced models based on presumed common pharmacophores,217–221 and the field has also been reviewed.222 IX. CLINICAL APPLICATIONS OF TAXOL AND DOCETAXEL The preceding discussions have provided a brief and somewhat selective overview of the chemistry and biology of taxol. For most nonscientists, however, the key question is, “How well does it work: What are the clinical benefits of taxol and its sister drug docetaxel to the cancer patient?” It is difficult to give an unqualified answer to this question, in part because treatment modalities are continuing to improve. The use of taxol in cancer therapy has recently been reviewed,223 and several reviews have also covered the use of taxol or docetaxel for the treatment of specific cancers.224–228 At present, the taxane drugs taxol and docetaxel are used for the treatment of breast, lung, and ovarian carcinomas and also for AIDS-related Kaposi’s sarcoma. The taxanes have proven to be among the most active agents in treating advanced metastatic breast cancer,225 but their benefits in treating early-stage breast cancer have only recently been evaluated. Two recent reviews have now documented their value, however. One reviewer states, “The evidence is now clear that taxanes added to standard adjuvant regimens … can improve outcomes for patients with breast cancer,”229 and a second systematic review of taxane versus nontaxane regimens for treatment of early breast cancer concludes, “The results of this systematic review support the use of taxanes as adjuvant chemotherapy for women with early breast cancer and involved lymph nodes.” In the studies summarized in this review, the 5-yr relapse-free survival rate was 74% for the control (nontaxane) group versus 79% for the taxane-treated group.230 In the lung cancer area docetaxel is the drug of choice for the treatment of advanced non–small cell lung cancer that is refractory to primary chemotherapy.226 A study to compare the efficacy of three different treatment regimens (cisplatin and gemcitabine, cisplatin and docetaxel, or carbo- platin and taxol) found that the response rates and survival did not differ significantly between the regimens.224 The significance of taxol as an anticancer drug was first discovered for ovarian cancer, and this remains an important target. A European–Canadian study confirmed early findings that the com- bination of taxol–cisplatin is superior to the previously used regimen of cyclophosphamide–cispl- atin,231 and that this is now the standard of care for women with advanced ovarian cancer. Copyright © 2005 CRC Press, LLC Although the taxanes are certainly not the wonder drugs they were at one time thought to be, they have nevertheless brought significant benefits to many cancer patients. The new analogs referred to in the following section promise real improvements in efficacy over taxol and docetaxel, and improvements in drug delivery will also play a major role in improving treatment. In these connections, the recent announcement of the successful phase III trial of the albumin nanoparticle- based formulations of taxol ABI-007232 shows that improved formulations can have a dramatic effect. The overall response rate for ABI-007 in patients with metastatic breast cancer was 33%, compared with 19% for taxol. It is thus clear that taxol and its analogs will continue to play an important role in cancer chemotherapy well into the twenty-first century. X. TAXOL ANALOGS IN CLINICAL TRIALS As described in section V, numerous taxol analogs have been prepared, and several have entered preclinical development or clinical trial. An extensive listing of taxol analogs in development is contained in an excellent recent review,116 and readers are referred to this review for a listing of taxol analogs in preclinical development. Six taxol analogs are currently in phase II clinical trial. They are the simple C7 acyl derivatives 7-DHA-taxol (Taxoprexin, V.C.1),88 7-hexanoyltaxol (V.C.2),88 the C7 thioether V.C.3 (BMS- 184476),89–91 the cyclopropyl derivative V.C.14 (RPR-109881A),233 the C4 carbonate V.C.16 (BMS- 188797),107 and the 7,10-dimethyl ether X.1 (TXD258).234 Four additional taxol analogs are in phase I clinical trial. These are ortataxel (V.A.10),56 TL-00139 (V.E.5),123 DJ-927 (V.E.6),124 and BMS-275183 (X.2). A further 23 taxol analogs are in preclinical development; their structures are given in the review cited above. XI. CONCLUSION The botanists who collected the bark of T. brevifolia in 1962 could not have foreseen the enormous consequences that would flow from that simple act. Providentially, their action initiated a chain of events that has led to the present situation, in which the one lead compound of taxol has not only yielded two currently approved clinical agents but led to the development of 33 compounds that are either in clinical trial or advanced preclinical development. It is certain that some of these compounds will advance to clinical use, and taxol will thus be joined by a number of related compounds in providing clinical anticancer agents. In addition, the mechanism of action of taxol, which was unique when it was first discovered, has led to the discovery of several new natural and synthetic compounds with the same mechanism, as described in section VIII. In a real sense, taxol can thus be viewed as the progenitor of an even larger number of potential clinical agents. It is almost a certainty that some of them will turn out to have significantly better clinical profiles than taxol itself, with significant life extensions and even cures in some cases. That botanical collection in 1962, coupled with the skill and expertise of numerous scientists, has thus made a huge contribution to cancer treatment and must surely rank as the most significant such collections ever made! ACKNOWLEDGMENTS John Donne wrote that “No man is an island,” and this is certainly true of all scientists who have worked on taxol, as we all build on the knowledge and results of others in the area. The author is particularly grateful to his colleagues at the National Cancer Institute, and especially to the late Matthew Suffness for his early support of work on taxol when few others believed that it would ever become a clinically used drug. More recently, Gordon Cragg and David Newman, the coeditors of this volume, and Yali Hallock have provided continued support and encouragement. Copyright © 2005 CRC Press, LLC The author’s work reported herein was supported by grants from BMS and the National Cancer Institute, most recently under grant CA 69571, and such support is gratefully acknowledged. The author also acknowledges with gratitude the collaboration of his colleagues Susan Bane (SUNY Binghamton), Jake Schaefer (Washington University), and Jim Snyder (Emory University) and of the talented graduate students and research associates whose names are recorded in the publications from

his group. REFERENCES 1. Wani, M.C. et al., Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, J. Am. Chem. Soc., 93, 2325, 1971. 2. Itokawa, H. Introduction, in Taxus: The Genus Taxus, Itokawa, H. and Lee, K.-H., Eds. Taylor & Francis, London, 2003, 1. 3. Schiff, P.B., Fant, J., and Horwitz, S.B., Promotion of microtubule assembly in vitro by taxol, Nature, 277, 665, 1979. 4. Weiss, R.B. et al., Hypersensitivity reactions from taxol, J. Clin. Oncol., 8, 1263, 1990. 5. Rose, W.C. Preclinical Antitumor Activity of Taxanes, in Taxol, Science and Applications, Suffness, M. Ed., CRC Press, Inc., Boca Raton, FL, 1995, chap. 8. 6. McGuire, W.P. et al., Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms, Ann. Intern. Med., 111, 273, 1989. 7. Holmes, F.A. et al., Phase II trial of taxol, an active drug in the treatment of metastatic breast cancer, J. Nat. Cancer Inst., 83, 1797, 1991. 8. Chase, M., Cancer drug may save many human lives — at cost of rare trees, The Wall Street Journal, 217 (69), A1, A8, 1991. 9. Chauvière G. et al., Analyse structurale et etude biochimique de produits isoles de l’if: Taxus baccata L. (Taxacees), C. R. Acad. Sci. Paris, Ser. II, 293, 501, 1981. 10. Denis, J.-N. et al., A highly efficient, practical approach to natural taxol, J. Am. Chem. Soc., 110, 5917, 1988. 11. Holton, R.A., Biediger, R.J., and Boatman, P.D. Semisynthesis of taxol and taxotere. In Taxol, Science and Applications, Suffness, M., Ed., CRC Press, Inc., Boca Raton, FL, 1995, chap. 5. 12. Guèritte-Voegelein, F. et al., Chemical studies of 10-deacetyl baccatin III. Hemisynthesis of taxol derivatives, Tetrahedron, 42, 4451, 1986. 13. Bissery, M.-C. et al., Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I: preclinical experience, Anti-Cancer Drugs, 6, 339, 1995. 14. Guèritte, F. General and recent aspects of the chemistry and structure-activity relationships of taxoids, Curr. Pharm. Design, 7, 1229, 2001. 15. Takeya, K.; Plant tissue culture of taxoids, in Taxus: The Genus Taxus, Itokawa, H. and Lee, K.-H., Eds., Taylor and Francis, London, 2003, chap. 5. 16. Anonymous. Sustaining a responsible pipeline of new products. Bristol-Myers Squibb 2003 Sustain- ability Report http://www.bms.com/static/ehs/report/data/s03p25.html, Accessed May 13, 2004. 17. Rohmer, M. et al., Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate, Biochem. J., 295, 517, 1993. 18. Eisenreich, W. et al., Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin, Proc. Natl. Acad. Sci. USA, 93, 6431, 1996. 19. Koepp, A.E. et al., Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the com- mitted step of taxol biosynthesis in Pacific yew, J. Biol. Chem., 270, 8686, 1995. 20. Hezari, M., Lewis, N.G., and Croteau, R., Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosyn- thesis, Arch. Biochem. Biophys., 322, 437, 1995. 21. Wildung, M.R. and Croteau, R., A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis, J. Biol. Chem., 271, 9201, 1996. 22. Hezari, M. et al., Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures, Arch. Biochem. Biophys., 337, 185, 1997. Copyright © 2005 CRC Press, LLC 23. Lin, X. et al., Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew, Biochemistry, 35, 2968, 1996. 24. Floss, H.G. and Mocek, U., Biosynthesis of taxol, in Taxol: Science and Applications, Suffness, M., Ed., CRC Press, Inc., Boca Raton, FL, 1995, chap. 7. 25. Hefner, J. et al., Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa- 4(20),11(12)-dien-5α-ol: the first oxygenation step in taxol biosynthesis, Chem. Biol., 3, 479, 1996. 26. Walker, K. et al., Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)- dien-5α-ol O-acetyl transferase that catalyzes the first acylation step of taxol biosynthesis, Arch. Biochem. Biophys., 364, 273, 1999. 27. Wheeler, A.L. et al., Taxol biosynthesis: differential transformations of taxadien-5α-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells, Arch. Biochem. Biophys., 390, 265, 2001. 28. Horiguchi, T. et al., Studies on taxol biosynthesis. Preparation of 5α-acetoxytaxa-4(20),11-dien- 2α,10β-diol derivatives by deoxygenation of a taxadiene tetra-acetate obtained from Japanese yew, Tetrahedron, 59, 267, 2003. 29. Chau, M. et al., Taxol biosynthesis: molecular cloning and characterization of a cytochrome P450 taxoid 7β-hydroxylase, Chem. Biol., 11, 663, 2004. 30. Jennewein, S. et al., Taxol biosynthesis: taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase, Proc. Natl. Acad. Sci. USA, 98, 13595, 2001. 31. Jennewein, S. et al., Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in taxol biosynthesis, Proc. Natl. Acad. Sci. USA, 101, 9149, 2004. 32. Fleming, P.E., Mocek, U., and Floss, H.G., Biosynthesis of taxoids. Mode of formation of the taxol side chain, J. Am. Chem. Soc., 115, 805, 1993. 33. Fleming, P.E. et al., Biosynthetic studies on taxol, Pure Appl. Chem., 66, 2045, 1994. 34. Fleming, P.E. et al., Biosynthesis of taxoids. Mode of attachment of the taxol side chain, J. Am. Chem. Soc., 116, 4137, 1994. 35. Zhong, J.-J., Plant cell culture for production of paclitaxel and other taxanes, J. Biosci. Bioeng., 94, 591, 2002. 36. Tabata, H., Paclitaxel production by plant-cell-culture technology. Adv. Biochem. Engin./Biotechnol., 7, 1, 2004. 37. Yukimune, Y. et al., Methyl jasmonate-induced overproduction of paclitaxol and baccatin III in Taxus cell suspension cultures, Nat. Biotechnol., 14, 1129, 1996. 38. Genexol. Genexol: The World’s Best Paclitaxel. http://www.genexol.com/eng/app//manufac- ture/default.asp. Accessed Feb. 21, 2005. 39. Stierle, A., Strobel, G., and Stierle, D., Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew, Science, 260, 214, 1993. 40. Li, J.Y. et al., The induction of taxol production in the endophytic fungus — Periconia sp. from Torreya grandifolia, J. Ind. Micro. Biotechnol., 20, 259, 1998. 41. Strobel, G. et al., Natural products from endophytic microorganisms, J. Nat. Prod., 67, 257, 2004. 42. Schiff, P. B. and Horwitz, S. B., Taxol stabilizes microtubules in mouse fibroblast cells, Proc. Natl. Acad. Sci. USA, 77, 1561, 1980 43. Jordan, M.A. and Wilson, L. Microtubules and actin filaments: dynamic targets for cancer chemo- therapy, Curr. Opin. Cell Biol., 10, 123, 1998. 44. Jordan, M.A. et al., Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations, Proc. Natl. Acad. Sci. USA, 90, 9552, 1993. 45. Long, B.H. and Fairchild, C.R., Paclitaxel inhibits progression of mitotic cells to G1 phase by interference with spindle formation without affecting other microtubule functions during anaphase and telephase, Cancer Res., 54, 4355, 1994. 46. Haldar, S., Chintapalli, J., and Croce, C. M., Paclitaxel induces Bcl-2 phosphorylation and death of prostate cancer cells, Cancer Res., 56, 1253, 1996. 47. Blagosklonny, M.V. et al., Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death, Cancer Res., 57, 130, 1997. 48. Rodi, D.J. et al., Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol- binding protein, J. Mol. Biol., 285, 197, 1999. Copyright © 2005 CRC Press, LLC 49. Carboni, J. M., Singh, C. and Tepper, M. A., Taxol and lipopolysaccharide activation of a murine macrophage cell line and induction of similar tyrosine phosphoproteins, J. Natl. Cancer. Inst. Monogr., 15, 95, 1993. 50. Blagosklonny, M.V. and Fojo, T., Molecular effects of paclitaxel: Myths and reality (a critical review), Int. J. Cancer, 83, 151, 1999. 51. Abal, M., Andreu, J.M., and Barasoain, I., Taxanes: Microtubule and centrosome targets, and cell cycle dependent mechanisms of action, Curr. Cancer Drug Targets, 3, 193, 2003. 52. Wang, X., Itokawa, H., and Lee, K.-H., Structure-activity relationships of taxoids, in Taxus: The Genus Taxus, Itokawa, H. and Lee, K.-H., Eds. Taylor & Francis, London, 2003, 298. 53. Kingston, D. G. I. et al., The chemistry of taxol and related taxoids, in Progress in the Chemistry of Organic Natural Products Vol. 84, Herz, W. Falk, H., and Kirby, G. W., Eds., Springer, Vienna, 2002, 53–224. 54. Kingston, D. G. I., Taxol, a molecule for all seasons, Chem. Commun., 867, 2001. 55. Ojima, I. et al., New generation taxoids and hybrids of microtubule-stabilizing anticancer agents, in ACS Symposium Series 796, I. Ojima, Altmann K.-H., Eds., American Chemical Society, Washington, DC, 2001, chap. 4. 56. Ojima, I. et al., Medicinal chemistry and chemical biology of new generation taxane antitumor agents, Life, 53, 269, 2002. 57. Guèritte, F., General and recent aspects of the chemistry and structure-activity relationships of taxoids, Curr. Pharmaceutical Design, 7, 1229, 2001. 58. Samaranayake, G., Neidigh, K.A., and Kingston, D.G.I., Modified taxols. 8. Deacylation and reacy- lation of baccatin III, J. Nat. Prod., 56, 884, 1993. 59. Harriman, G.C.B. et al., The chemistry of the taxane diterpene: stereoselective synthesis of 10- deacetoxy-11,12-epoxypaclitaxel, Tetrahedron Lett., 36, 8909, 1995. 60. Chen, S.-H. et al., Taxol structure-activity relationships: synthesis and biological evaluation of 10- deoxytaxol, J. Org. Chem., 58, 2927, 1993. 61. Kelly, R.C. et al., 12-13-Isobaccatin III. Taxane enol esters (12,13-isotaxanes), J. Am. Chem. Soc., 118, 919, 1996. 62. Uoto, K. et al., First synthesis and cytotoxic activity of novel docetaxel analogs modified at the C- 18-position, Bioorg. Med. Chem. Lett., 7, 2991, 1997. 63. Samaranayake, G. et al., Modified taxols. 5. Reaction of taxol with electrophilic reagents, and prepa- ration of a rearranged taxol derivative with tubulin assembly activity, J. Org. Chem., 56, 5114, 1991. 64. Appendino, G. et al., 14β-Hydroxy-10-deacetylbaccatin III, a new taxane from Himalayan yew (Taxus wallichiana Zucc.), J. Chem. Soc., Perkin Trans., 1, 21, 2925, 1992. 65. Ojima, I. et al., Structure-activity relationships of new taxoids derived from 14β-hydroxy-10-deacetyl- baccatin III, J. Med. Chem., 37, 1408, 1994. 66. Ojima, I. et al., Syntheses and structure-activity relationships of novel nor-seco taxoids, J. Org. Chem., 63, 1637, 1998. 67. Johnson, R.A. et al., Taxol chemistry. 7-O-triflates as precursors to olefins and cyclopropanes, Tetra- hedron Lett., 35, 7893, 1994. 68. Zheng, Q.Y. et al., Deacetylation of paclitaxel and other taxanes, Tetrahedron Lett., 36, 2001, 1995. 69. Holton, R.A., Somoza, C., and Chai, K.-B., A simple synthesis of 10-deacetoxytaxol derivatives, Tetrahedron Lett., 35, 1665, 1994. 70. Georg, G.I. and Cheruvallath, Z.S., Samarium diiodide-mediated deoxygenation of taxol: a one-step synthesis of 10-deacetoxytaxol, J. Org. Chem., 59, 4015, 1994. 71. Chaudhary, A.G. and Kingston, D.G.I., Synthesis of 10-deacetoxytaxol and 10-deoxytaxotere, Tetra- hedron Lett., 34, 4921, 1993. 72. Ojima, I. et al., Syntheses and structure-activity relationships of the second-generation antitumor taxoids: exceptional activity against drug-resistant cancer cells, J. Med. Chem., 39, 3889, 1996. 73. Klein, L.L., Synthesis of 9-dihydrotaxol: a novel bioactive taxane, Tetrahedron Lett., 34, 2047, 1993. 74. Li, L. et al., Synthesis and biological evaluation of C-3-modified analogs of 9(R)-dihydrotaxol, J. Med. Chem., 37, 2655, 1994. 75. Klein, L.L. et al., Antitumor activity of 9(R)-dihydrotaxane analogs, J. Med. Chem., 38, 1482, 1995. 76. Klein, L. L., Yeung, C. M., and Li, L., 9-Deoxotaxanes, U.S. patent 5,440,056, August 8, 1995. Copyright © 2005 CRC Press, LLC 77. Chen, S.-H., Wei, J.-M., and Farina, V., Taxol structure-activity relationships: Synthesis and biological evaluation of 2-deoxytaxol, Tetrahedron Lett., 34, 3205, 1993. 78. Chaudhary, A.G., Chordia, M.D., and Kingston, D.G.I., A novel benzoyl group migration: synthesis and biological evaluation of 1-benzoyl-2-des(benzoyloxy)paclitaxel, J. Org. Chem., 60, 3260, 1995. 79. Chordia, M.D. and Kingston, D.G.I., Synthesis and biological evaluation of 2-epi-paclitaxel, J. Org. Chem., 61, 799, 1996. 80. Fang, W.-S., Fang, Q.-C., and Liang, X.-T., Synthesis of the 2α-benzoylamido analogue of docetaxel, Tetrahedron Lett., 42, 1331, 2001. 81. Chaudhary, A.G. et al., Unexpectedly facile hydrolysis of the 2-benzoate group of taxol and synthesis of analogs with increased activities, J. Am. Chem. Soc., 116, 4097, 1994. 82. Kingston, D.G.I. et al., Synthesis and biological evaluation of 2-acyl analogues of paclitaxel (taxol), J. Med. Chem., 41, 3715, 1998. 83. Nicolaou, K.C. et al., Chemical synthesis and biological evaluation of C-2 taxoids, J. Am. Chem. Soc., 117, 2409, 1995. 84. Georg, G.I. et al., Synthesis of 2-O-heteroaroyl taxanes: Evaluation of microtubule assembly promo- tion and cytotoxicity, Bioorg. Med. Chem. Lett., 5, 115, 1995. 85. Ojima, I. et al., Syntheses and structure-activity relationships of nonaromatic taxoids: Effects of alkyl and alkenyl ester groups on cytotoxicity, J. Med. Chem., 40, 279, 1997. 86. Kingston, D.G.I., Chordia, M.D., and Jagtap, P.G., Synthesis and biological evaluation of 1-deoxy- paclitaxel analogues, J. Org. Chem., 64, 1814, 1999. 87. Bradley, M. O.

et al., Preparation of conjugates of all-cis-docosahexaenoic acid and paclitaxel as antitumor agents. PCT Int. Appl. WO 9,744,336, 1997. 88. Ali, S. et al., Hydrolyzable hydrophobic taxanes: synthesis and anti-cancer activities, Anti-Cancer Drugs, 12, 117, 2001. 89. Altstadt, T.J. et al., Synthesis and antitumor activity of novel C-7 paclitaxel ethers: discovery of BMS- 184476, J. Med. Chem., 44, 4577, 2001. 90. Ojima, I. and Geney, R., BMS-184476 (Bristol-Myers Squibb), Curr. Opin. Invest. Drugs, 4, 732, 2003. 91. Plummer, R. et al., Phase I and pharmacokinetic study of the new taxane analog BMS-184476 given weekly in patients with advanced malignancies, Clin. Cancer Res., 8, 2788, 2002. 92. Magri, N.F. and Kingston, D.G.I., Modified taxols. 2. Oxidation products of taxol, J. Org. Chem., 51, 797, 1986. 93. Chaudhary, A.G., Rimoldi, J.M., and Kingston, D.G.I., Modified taxols. 10. Preparation of 7-deox- ytaxol, a highly bioactive taxol derivative, and interconversions of taxol and 7-epi-taxol, J. Org. Chem., 58, 3798, 1993. 94. Chen, S.-H. et al., Synthesis of 7-deoxy- and 7,10-dideoxytaxol via radical intermediates, J. Org. Chem., 58, 5028, 1993. 95. Poujol, H. et al., Taxoides: 7-deshydroxy-10-acetyldocetaxel et nouveaux analogues prepares a partir des alcaloides de l’if, Tetrahedron, 53, 12575, 1997. 96. Johnson, R.A. et al., Taxol chemistry. 7-O-triflates as precursors to olefins and cyclopropanes, Tetra- hedron Lett., 35, 7893, 1994. 97. Liang, X. et al., Synthesis and biological evaluation of paclitaxel analogs modified in ring C, Tetra- hedron Lett., 36, 2901, 1995. 98. Yuan, H. and Kingston, D.G.I., Synthesis of 6α-hydroxypaclitaxel, the major human metabolite of paclitaxel, Tetrahedron Lett., 39, 4967, 1998. 99. Wittman, M.D., Kadow, J.F., and Vyas, D.M., Stereospecific synthesis of the major human metabolite of paclitaxel, Tetrahedron Lett., 41, 4729, 2000. 100. Liang, X. et al., Paclitaxel analogs modified in ring C: Synthesis and biological evaluation, Tetrahe- dron, 53, 3441, 1997. 101. Chen, S.-H. et al., Serendipitous synthesis of a cyclopropane-containing taxol analog via anchimeric participation of an unactivated angular methyl group, J. Org. Chem., 58, 4520, 1993. 102. Bouchard, H. et al., Improved access to 19-nor-7β,8β-methylene-taxoids and formation of a 7- membered C-ring analog of docetaxel by electrochemistry, Tetrahedron Lett., 35, 9713, 1994. 103. Appendino, G. et al., Synthesis and evaluation of C-seco paclitaxel analogues, Tetrahedron Lett., 38, 4273, 1997. Copyright © 2005 CRC Press, LLC 104. Pratesi, G. et al., IDN 5390: an oral taxane candidate for protracted treatment schedules, Br. J. Cancer, 88, 965, 2003. 105. Appendino, G. et al., Structure-activity relationships of ring C-secotaxoids. 1. Acylative modifications, J. Nat. Prod., 67, 184, 2004. 106. Neidigh, K.A. et al., Synthesis and biological evaluation of 4-deacetylpaclitaxel, Tetrahedron Lett., 35, 6839, 1994. 107. Chen, S.-H., Discovery of a novel C-4 modified 2nd generation paclitaxel analog BMS-188797, Frontiers Biotechnol. Pharmaceut., 3, 157, 2002. 108. Mastalerz, H. et al., The discovery of BMS-275183: an orally efficacious novel taxane, Bioorg. Med. Chem. 11, 4315, 2003. 109. Chordia, M.D. et al., Synthesis and bioactivity of 2,4-diacyl analogues of paclitaxel, Bioorg. Med. Chem., 9, 171, 2001. 110. Marder-Karsenti, R. et al., Synthesis and biological evaluation of D-ring-modified taxanes: 5(20)- azadocetaxel analogs, J. Org. Chem., 62, 6631, 1997. 111. Gunatilaka, A.A.L. et al., Synthesis and biological evaluation of novel paclitaxel (taxol) D-ring modified analogues, J. Org. Chem., 64, 2694, 1999. 112. Wang, M. et al., The oxetane ring in taxol, J. Org. Chem., 65, 1059, 2000. 113. Dubois, J. et al., Synthesis of 5(20)deoxydocetaxel, a new active docetaxel analogue, Tetrahedron Lett., 41, 3331, 2000. 114. Deka, V. et al., Deletion of the oxetane ring in docetaxel analogues: synthesis and biological evaluation, Org. Lett., 5, 5031, 2003. 115. Barboni, L. et al., Novel D-seco paclitaxel analogues: synthesis, biological evaluation, and model testing, J. Org. Chem., 66, 3321, 2001. 116. Cragg, G.M. and Newman, D.J., A tale of two tumor targets: topoisomerase I and tubulin. The Wall and Wani contribution to cancer chemotherapy, J. Nat. Prod., 67, 232, 2004. 117. He, L. et al., A common pharmacophore for taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain, Biochemistry, 39, 3972, 2000. 118. Swindell, C.S. et al., Biologically active taxol analogues with deleted A-ring side chain substituents and variable C-2 configurations, J. Med. Chem., 34, 1176, 1991. 119. Guèritte-Voegelein, F. et al., Relationships between the structure of taxol analogues and their antim- itotic activity, J. Med. Chem., 34, 992, 1991. 120. Barboni, L. et al., Synthesis and NMR-driven conformational analysis of taxol analogues conforma- tionally constrained on the C13 side chain, J. Med. Chem., 44, 1576, 2001. 121. Jayasinghe, L.R. et al., Structure-activity studies of antitumor taxanes: synthesis of novel C-13 side chain homologated taxol and taxotere analogs, J. Med. Chem., 37, 2981, 1994. 122. Georg, G.I. et al., Synthesis, conformational analysis, and biological evaluation of heteroaromatic taxanes, J. Org. Chem., 61, 2664, 1996. 123. Sampath, D. et al., MAC-321, a novel taxane with greater efficacy than paclitaxel and docetaxel in vitro and in vivo, Mol. Cancer Ther., 873, 2003. 124. Shionoya, M. et al., DJ-927, a novel oral taxane, overcomes P-glycoprotein-mediated multidrug resistance in vitro and in vivo, Cancer Sci. 94, 459, 2003. 125. Rose, W.C. et al., Preclinical pharmacology of BMS-275183, an orally active taxane, Clin. Cancer Res., 7, 2016, 2001. 126. Magri, N.F. and Kingston, D.G.I., Modified taxols. 4. Synthesis and biological activity of taxols modified in the side chain, J. Nat. Prod., 51, 298, 1988. 127. Denis, J.-N. et al., Docetaxel (taxotere) derivatives: novel NbCl3-based stereoselective approach to 2- methyldocetaxel, J. Chem. Soc. Perkin Trans. 1, 1811, 1995. 128. Kant, J. et al., Diastereoselective addition of Grignard reagents to azetidine-2,3-dione: Synthesis of novel taxol analogues, Tetrahedron Lett., 37, 6495, 1996. 129. Ojima, I., Wang, T., and Delaloge, F., Extremely stereoselective alkylation of 3-siloxy-β-lactams and its applications to the asymmetric syntheses of novel 2-alkylisoserines, their dipeptides, and taxoids, Tetrhedron Lett., 39, 3663, 1998. 130. Battaglia, A. et al., Synthesis and biological evaluation of 2-methyl taxoids derived from baccatin III and 14β-OH-baccatin II 1,14-carbonate, J. Med. Chem., 46, 4822, 2003. Copyright © 2005 CRC Press, LLC 131. Qi, X. et al., Synthesis of novel thiol surrogate of taxol: 2-deoxy-2-mercaptopaclitaxel, Tetrahedron, 60, 3599, 2004. 132. Uoto, K. et al., Synthesis and structure-activity relationships of novel 2,2-difluoro analogues of docetaxel, Chem. Pharm. Bull., 45, 1793, 1997. 133. Yamaguchi, T. et al., Synthesis of taxoids 5. Synthesis and evaluation of novel water-soluble prodrugs of a 3-desphenyl-3-cyclopropyl analogue of docetaxel, Bioorg. Med. Chem. Lett., 9, 1639, 1999. 134. Georg, G.I. et al., The medicinal chemistry of taxol, in Taxol: Science and Applications, Suffness, M., Ed., CRC Press, Inc., Boca Raton, FL, 1995, chap. 13. 135. Deutsch, H.M. et al., Synthesis of congeners and prodrugs. 3. Water-soluble prodrugs of taxol with potent antitumor activity, J. Med. Chem., 32, 788, 1989. 136. Zhao, Z., Kingston, D.G.I., and Crosswell, A.R., Modified taxols, 6. Preparation of water-soluble prodrugs of taxol, J. Nat. Prod., 54, 1607, 1991. 137. Mathew, A.E. et al., Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity, J. Med. Chem., 35, 145, 1992. 138. Ueda, Y. et al., Novel water soluble phosphate prodrugs of taxol possessing in vivo antitumor activity, Bioorg. Med. Chem. Lett., 3, 1761, 1993. 139. Hayashi, Y. et al., A novel approach of water-soluble paclitaxel prodrug with no auxiliary and no byproduct: design and synthesis of isotaxel, J. Med. Chem., 46, 3782, 2003. 140. Skwarczynski, M. et al., O-N intramolecular acyl migration strategy in water-soluble prodrugs of taxoids, Bioorg. Med. Chem., 13, 4441, 2003. 141. Leenders, R.G.G. et al., β-glucuronyl carbamate based pro-moieties designed for prodrugs in ADEPT, Tetrahedron Lett., 36, 1701, 1995. 142. de Bont, D.B.A. et al., Synthesis and biological activity of β-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT, Bioorg. Med. Chem. 5, 405, 1997. 143. Schmidt, F. et al., Cancer chemotherapy: a paclitaxel prodrug for ADEPT (antibody-directed enzyme prodrug therapy), Eur. J. Org. Chem., 2129, 2001. 144. Bouvier, E. et al., First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT, Biorg. Med. Chem., 12, 969, 2004. 145. de Groot, F.M.H. et al., Synthesis and biological evaluation of 2-carbamate-linked and 2-carbonate- linked prodrugs of paclitaxel: selective activation by the tumor-associated protease plasmin, J. Med. Chem., 43, 3093, 2000. 146. Takahashi, T., Tsukamoto, H., and Yamada, H., Design and synthesis of a water-soluble taxol analog: taxol-sialyl conjugate, Bioorg. Med. Chem. Lett., 8, 113, 1998. 147. Luo, Y. and Prestwich, G.D., Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate, Bioconjugate Chem., 10, 755, 1999. 148. Bradley, M.O. et al., Tumor targeting by conjugation of DHA to paclitaxel, J. Controlled Release, 74, 233, 2001. 149. Bradley, M.O. et al., Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel, Clin. Cancer Res., 7, 3229, 2001. 150. Lee, J.W. et al., Synthesis and evaluation of taxol-folic acid conjugates as targeted antineoplastics, Bioorg. Med. Chem., 10, 2397, 2002. 151. Kirschberg, T.A. et al., Arginin-based molecular transporters: the synthesis and chemical evaluation of releasable taxol-transporter conjugates, Org. Lett., 5, 3459, 2003. 152. Guillemard, V. and Saragovi, H.U., Taxane-antibody conjugates afford potent cytotoxicity, enhanced solubility, and tumor target selectivity, Cancer Res., 61, 694, 2001. 153. Ojima, I. et al., Tumor-specific novel taxoid-monoclonal antibody conjugates, J. Med. Chem., 45, 5620, 2002. 154. Wuts, P.G.M., Semisynthesis of taxol, Curr. Opin. Drug Disc. Dev., 1, 329, 1998. 155. Ojima, I. et al., New and efficient approaches to the semisynthesis of taxol and its C-13 side chain analogs by means of β-lactam synthon method, Tetrahedron, 48, 6985, 1992. 156. Ojima, I. et al., Efficient and practical asymmetric synthesis of the taxol C-13 side chain, N-benzoyl- (2R,3S)-3-phenylisoserine, and its analogues via chiral 3-hydroxy-4-aryl-β-lactams through chiral ester enolate-imine cycl, J. Org. Chem., 56, 1681, 1991. Copyright © 2005 CRC Press, LLC 157. Georg, G.I. et al., An efficient semisynthesis of taxol from (3R,4S)-N-benzoyl-3-[(t-butyldimethylsi- lyl)oxy]-4-phenyl-2-azetidinone and 7-(triethylsilyl)baccatin III, Bioorg. Med. Chem. Lett., 3, 2467, 1993. 158. Palomo, C. et al., Asymmetric synthesis of α-keto β-lactams via [2+2] cycloaddition reaction — a concise approach to optically active α-hydroxy β-lactams and β-alkyl(aryl)isoserines, Tetrahedron Lett., 34, 6325, 1993. 159. Holton, R.A. and Liu, J.H., A novel asymmetric synthesis of cis-3-hydroxy-4-aryl azetidin-2-ones, Bioorg. Med. Chem. Lett., 3, 2475, 1993. 160. Farina, V., Hauck, S.I., and Walker, D.G., A simple chiral synthesis of the taxol side chain, Synlett, 1, 761, 1992. 161. Bourzat, J.D. and Commercon, A., A practical access to chiral phenylisoserinates, preparation of taxotere analogs, Tetrahedron Lett., 34, 6049, 1993. 162. Shimizu, M., Ishida, T., and Fujisawa, T., Highly stereocontrolled construction of 3-alkoxyazetidin- 2-ones using ester enolate-imine condensation, Chem. Lett., 1403, 1994. 163. Song, C.E. et al., A new synthetic route to (3R,4S)-3-hydroxy-4-phenylazetidin-2-one as a taxol side chain precursor, Tetrahedron: Asymmetry, 9, 983, 1998. 164. Brieva, R., Crich, J.Z., and Sih, C.J., Chemoenzymatic synthesis of the C-13 side chain of taxol: optically-active 3-hydroxy-4-phenyl β-lactam derivatives, J. Org. Chem., 58, 1068, 1993. 165. Lin, S. et al., Synthesis of highly potent second-generation taxoids through effective kinetic resolution coupling of racemic β-lactams with baccatins, Chirality, 12, 431, 2000. 166. Kingston, D.G.I. et al., Synthesis of taxol from baccatin III via an oxazoline intermediate, Tetrahedron Lett., 35, 4483, 1994. 167. Gennari, C. et al., Taxol semisynthesis: a highly enantio- and diastereoselective synthesis of the side chain and a new method for ester formation at C-13 using thioesters, J. Org. Chem., 62, 4746, 1997. 168. Gennari, C. et al., Semisynthesis of taxol: A highly enantio- and diastereoselective synthesis of the side chain and a new method for ester formation at C13 using thioesters, Angew. Chem. Int. Ed. Engl., 35, 1723, 1996. 169. Yamaguchi, T. et al., Synthesis of taxoids 4. Novel and versatile methods for preparation of new taxoids by employing cis- or trans-phenyl glycidic acid, Tetrahedron, 55, 1005, 1999. 170. Mandai, T. et al., A semisynthesis of paclitaxel via a 10-deacetylbaccatin III derivative bearing a β- keto ester appendage, Tetrahedron Lett., 41, 243, 2000. 171. Xiao, Z., Itokawa, H., Lee, and K.-H., Total synthesis of taxoids, in Taxus: The Genus Taxus, Itokawa, H. and Lee, K.-H., Eds., Taylor & Francis, London, 2003, chap. 9. 172. Holton, R.A. et al., First total synthesis of taxol. 1. Functionalization of the B ring, J. Am. Chem. Soc., 116, 1597, 1994. 173. Holton, R. A. et al., First total synthesis of taxol. 2. Completion of the C and D rings, J. Am. Chem. Soc., 116, 1599, 1994. 174.

Nicolaou, K.C. et al., Total synthesis of taxol, Nature, 367, 630, 1994. 175. Danishefsky, S.J. et al., Total synthesis of baccatin III and taxol, J. Am. Chem. Soc., 118, 2843, 1996. 176. Wender, P.A. et al., The pinene path to taxanes. 5. Stereocontrolled synthesis of a versatile taxane precursor, J. Am. Chem. Soc., 119, 2755, 1997. 177. Wender, P.A. et al., The pinene path to taxanes. 6. A concise stereocontrolled synthesis of taxol, J. Am. Chem. Soc., 119, 2757, 1997. 178. Kusama, H. et al., Enantioselective total synthesis of (–)-taxol, J. Am. Chem. Soc., 122, 3811, 2000. 179. Mukaiyama, T. et al., Asymmetric total synthesis of taxol, Chem. Eur. J., 5, 121, 1999. 180. Rao, S. et al., 3-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of β-tubulin, J. Biol. Chem., 269, 3132, 1994. 181. Rao, S. et al., Characterization of the taxol binding site on the microtubule, J. Biol. Chem., 270, 20235, 1995. 182. Rao, S. et al., Characterization of the taxol binding site on the microtubule, J. Biol. Chem., 274, 37990, 1999. 183. Dasgupta, D. et al., Synthesis of a photoaffinity taxol analogue and its use in labeling tubulin, J. Med. Chem., 37, 2976, 1994. Copyright © 2005 CRC Press, LLC 184. Loeb, C. et al., [3H](azidophenyl)ureido taxoid photolabels peptide amino acids 281–304 of β-tubulin, Biochemistry, 36, 3820, 1997. 185. Lowe, J. et al., Refined structure of β-tubulin at 3.5 Å resolution, J. Mol. Biol., 313, 1045, 2001. 186. Nogales, E., Wolf, S.G., and Downing, K.H., Structure of the αβ-tubulin dimer by electron crystal- lography, Nature, 39, 199, 1998. 187. Snyder, J.P. et al., The binding conformation of taxol in β tubulin: a model based on the electron crystallographic density, Proc. Natl. Acad. Sci. USA, 98, 5312, 2001. 188. Ojima, I., Inoue, T., and Chakravarty, S., Enantiopure fluorine-containing taxoids: potent anticancer agents and versatile probes for biomedical problems, J. Fluorine Chem., 97, 3, 1999. 189. Li, Y. et al., Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR, Biochemistry, 39, 281, 2000. 190. Dubois, J. et al., Conformation of taxotere and analogues determined by NMR spectroscopy and molecular modeling studies, Tetrahedron, 49, 6533, 1993 191. Williams, H.J. et al., NMR and molecular modeling study of the conformations of taxol and of its side chain methylester in aqueous and non-aqueous solution, Tetrahedron, 49, 6545, 1993. 192. Cachau, R.E. et al., Solution structure of taxol determined using a novel feedback-scaling procedure for NOE-restrained molecular dynamics, Supercomputer Appl. High Performance Comput., 8, 24, 1994. 193. Vander Velde, D.G. et al., “Hydrophobic collapse” of taxol and taxotere solution conformations in mixtures of water and organic solvent, J. Am. Chem. Soc., 115, 11650, 1993. 194. Paloma, L.G. et al., Conformation of a water-soluble derivative of taxol in water by 2D-NMR spectroscopy, Chem. Biol., 1, 107, 1994. 195. Ojima, I. et al., A novel approach to the study of solution structures and dynamic behavior of paclitaxel and docetaxel using fluorine-containing analogs as probes, J. Am. Chem. Soc., 119, 5519, 1997. 196. Snyder, J.P. et al., The conformations of taxol in chloroform, J. Am. Chem. Soc., 122, 724, 2000. 197. He, L. et al., A common pharmacophore for taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain, Biochemistry, 39, 3972, 2000. 198. Boge, T.C. et al., Conformationally restricted paclitaxel analogues: Macrocyclic mimics of the “hydro- phobic collapse” conformation, Bioorg. Med. Chem. Lett., 9, 3047, 1999. 199. Ojima, I. et al., A common pharmacophore for cytotoxic natural products that stabilize microtubules, Proc. Natl. Acad. Sci. USA, 96, 4256, 1999. 200. Querolle, O. et al., Synthesis of novel macrocyclic docetaxel analogues. Influence of their macrocyclic ring size on tubulin activity, J. Med. Chem., 46, 3623, 2003. 201. Ganesh, T. et al., The bioactive taxol conformation of β-tubulin: experimental evidence from highly active constrained analogs, Proc. Natl. Acad. Sci. USA, 101, 10006, 2004. 202. Gerth, K. et al., Antibiotics from gliding bacteria. 74. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria): production, physico-chemical and biological properties, J. Antibiot., 49, 560, 1996. 203. Bollag, D.M. et al., Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action, Cancer Res., 55, 2325, 1995. 204. ter Haar, E. et al., Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol, Biochemistry, 35, 243, 1996. 205. Lindel, T. et al., Eleutherobin, a new cytotoxin that mimics paclitaxel (taxol) by stabilizing microtu- bules, J. Am. Chem. Soc., 119, 8744, 1997. 206. Dupont, C. et al., D-ring substituted rhazinilam analogues: semisynthesis and evaluation of antitubulin activity, Bioorg. Med. Chem., 7, 2961, 1999. 207. Mooberry, S.L. et al., Laulimalide and isolaulimalide, new paclitaxel-like microtubule-stabilizing agents, Cancer Res., 59, 653, 1999. 208. Vanderwal, C.D. et al., Postulated biogenesis of WS9885B and progress toward an enantioselective synthesis, Org. Lett., 1, 645, 1999. 209. Roux, D. et al., Structure-activity relationship of polyisoprenyl benzophenones from Garcinia pyrifera on the tubulin/microtubule system, J. Nat. Prod., 63, 1070, 2000. 210. Miglietta, A. et al., Biological properties of jatrophane polyesters, new microtubule-interacting agents, Cancer Chemother. Pharmacol., 51, 67, 2003. Copyright © 2005 CRC Press, LLC 211. Isbrucker, R.A. et al., Tubulin polymerizing activity of dictyostatin-1, a polyketide of marine sponge origin, Biochem. Pharmacol., 66, 75, 2003. 212. Wang, Z. et al., Synthesis of B-ring homologated estradiol analogues that modulate tubulin polymer- ization and microtubule stability, J. Med. Chem., 43, 2419, 2000. 213. Madari, H. et al., Dicoumarol: a unique microtubule stabalizing natural product that is synergistic with taxol, Cancer Res., 63, 1214, 2003. 214. Fojo, T. and Giannakakou, P., Taxol and other microtubule-interactive agents, Curr. Opin. Oncol. Endocrine Metabol. Investig. Drugs, 2, 293, 2000. 215. Altmann, K.-H., Microtubule-stabilizing agents: a growing class of important anticancer drugs, Curr. Opin. Chem. Biol., 5, 424, 2001. 216. Myles, D.C., Emerging microtubule stabilizing agents for cancer chemotherapy, Ann. Rep. Med. Chem., 37, 125, 2002. 217. Jimenez-Barbero, J., Amat-Guerri, F., and Snyder, J.P., The solid state, solution and tubulin-bound conformations of agents that promote microtubule stabilization, Curr. Med. Chem. Anti-Cancer Agents, 2, 91, 2002. 218. Manetti, F. et al., 3D QSAR studies of the interaction between β-tubulin and microtubule stabilizing antimitotic agents (msaa). A combined pharmacophore generation and pseudoreceptor modeling approach applied to taxanes and epothilones, Il Farmaco, 58, 357, 2003. 219. Winkler, J.D. and Axelsen, P.H., A model for the taxol (paclitaxel)/epothilone pharmacophore, Bioorg. Med. Chem. Lett., 6, 2963, 1996. 220. Giannakakou, P. et al., A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells, Proc. Natl. Acad. Sci. USA, 97, 2904, 2000. 221. Wang, M. et al., A unified and quantitative receptor model for the microtubule binding of paclitaxel and epothilone, Org. Lett., 1, 43, 1999. 222. He, L., Orr, G.A., and Horwitz, S.B., Novel molecules that interact with microtubules and have functional activity similar to taxol, Drug Discov. Today, 6, 1153, 2001. 223. Mekhail, T. and Markman, M., Paclitaxel in cancer therapy, Expert Opin. Pharmacother., 3, 755, 2002. 224. Levin, M., The role of taxanes in breast cancer treatment, Drugs Today, 37, 57, 2001. 225. Rowinsky, E.K. The development and clinical utility of the taxane class of antimicrotubule chemo- therapy agents. Ann. Rev. Med. 48, 353, 1997. 226. Kris, M.G. and Manegold, C., Docetaxel (taxotere) in the treatment of non–small cell lung cancer: an international update, Sem. Oncol., 28, 1, 1, 2001. 227. Michaud, L.B., Valero, V., and Hortobagyi, G., Risks and benefits of taxanes in breast and ovarian cancer, Drug Safety, 23, 401, 2000. 228. Calderoni, A. and Cerny, T., Taxanes in lung cancer: a review with focus on the European experience, Crit. Rev. Oncol./Hematol., 38, 105, 2001. 229. Hudis, C., The use of taxanes in early breast cancer, EJC Suppl., 1, 1, 2003. 230. Nowak, A.K. et al., Systematic review of taxane-containing versus non-taxane-containing regimens for adjuvant and neoadjuvant treatment of early breast cancer, Lancet Oncol., 5, 372, 2004. 231. Piccart, M.J. et al., Randomized intergroup trial of cisplatin-paclitaxel versus cisplatin-cyclophospha- mide in women with advanced epithelial ovarian cancer: three-year results, J. Natl. Cancer Inst., 92, 699, 2000. 232. Garber, K., Improved paclitaxel formulation hints at new chemotherapy approach, J. Natl. Cancer Inst., 96, 90, 2004. 233. Ojima, I. and Geney, R., 109881 (Aventis), Curr. Opin. Investig. Drugs, 4, 737, 2003. 234. Cisternino, S., et al., Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood-brain barrier in mice and rats. Br. J. Pharmacol. 138, 1367, 2003. Copyright © 2005 CRC Press, LLC 7 The Vinca Alkaloids‡ Françoise Guéritte and Jacques Fahy CONTENTS I. Introduction II. Discovery of the Antitumor Vinca Alkaloids III. Mechanism of Action IV. Semisynthesis and Total Synthesis of Vinca Alkaloids V. Medicinal Chemistry VI. Clinical Applications VII. Conclusions References I. INTRODUCTION Vinca alkaloids are a family of indole–indoline dimeric compounds coming from the genus Apo- cynaceae, and they represent one of the most important classes of anticancer agents. Vinca alkaloids became clinically useful in cancer chemotherapy after their discovery in the late 1950s. More than 40 years later, the two natural drugs vinblastine and vincristine are still widely used in cancer chemotherapy, and semisynthetic analogs, such as vindesine and vinorelbine, have been developed after intensive synthetic and structure–activity relationship studies. Other analogs have emerged as potentially potent drugs, and some of these, such as vinflunine, will certainly enrich the family of anticancer vinca alkaloids in the near future. This review summarizes current knowledge about the vinca alkaloids, from their discovery to clinical applications, with an emphasis on the latest analogs that may be of interest in cancer chemotherapy. II. DISCOVERY OF THE ANTITUMOR VINCA ALKALOIDS1 Catharanthus roseus (L.) G. Don (formerly named as Vinca rosea L.) is known as the Madagascar periwinkle and belongs to the Apocynaceae family. This perennial plant has dark green leaves and pink or white flowers. It is cultivated in most warm countries as an ornamental in parks and gardens, and many varieties have been developed with various colors. In folkloric medicine, leaves of C. roseus were used to treat various diseases, and investigators from different countries of the world have reported hypotensive, hypoglycemic, and purgative properties for this plant.2 The antitumor properties of C. roseus were discovered independently in the 1950s by two teams, one Canadian and one American. In the early 1950s, Clark Noble, one of the first members of the University of Toronto insulin team, sent C. roseus leaves used to treat diabetes in Jamaica to his brother, Robert Noble. The challenge for Noble, working in Collip’s laboratory at the University of Western Ontario, was to find substances that could affect blood glucose levels. Instead of these properties, Noble found the leaves to have a strong effect on white blood cell counts and bone marrow. Together with the chemist Charles T. Beer, he ‡ This review is dedicated to Professor Pierre Potier, whose contribution in the field of Vinca alkaloids led to the discovery of new antitumor drugs. Copyright © 2005 CRC Press, LLC 11' 8 7' OH OH ' 12' 21' C' N N 4' 20' 13' A' 1' D' B' 18' 3' 7 9 14' N 19' 10 N 6 N N H 14 21 H 11 H3CO2C 20 H3CO2C H 4 H H CO OCOCH H 3 3 3CO 1 3 OCOCH3 17 N H N H CO2CH3 HO CO2CH3 CH OH CHO 3 1 2 FIGURE 7.1 Vinblastine (1) and Vincristine (2). isolated the active principle, which they named vincaleukoblastine; the name was later changed to vinblastine (1).3 Other investigations by Svoboda, Johnson, and collaborators at Lilly Laboratories, based on the reproducible antitumor activity of extracts of C. roseus leaves, resulted in the isolation of vinblastine.4 These studies also led to the isolation of other bioactive alkaloids, including leurocris- tine, also named vincristine 2, which was isolated from the leaves of the periwinkle.5 Vinblastine 1 and vincristine 2 are isolated in minute amounts from the leaves of C. roseus (Figure 7.1). For this reason, the semisynthesis and total synthesis of these binary alkaloids has been the subject of a number of studies, as we will see later on. Since the initial clinical trials in the 1960s, the so-called vinca alkaloids, vinblastine (Velban® or Velbe®) and vincristine (Oncovin®), have been widely used in the treatment of different types of cancer.

The complex structure and stereochemistry of vinblastine 1 and vincristine 2 were elegantly established by Neuss and his colleagues6 and by Moncrief and Lipscomb.7 Both vinblastine and vincristine are dimeric compounds possessing an indole moiety (the velbanamine “upper” part) and a vindoline dihydroindole nucleus. They differ by the substituent attached to the nitrogen of the dihy- droindole part, with vinblastine bearing an N-methyl group and vincristine possessing an N-formyl function. Two systems have been adopted to number the bisindole-type alkaloids: biogenetic and International Union of Pure and Applied Chemistry numbering.1 The latter will be used throughout this review. Despite their small structural differences, vinblastine 1 and vincristine 2 differ strongly in their antitumor properties and clinical toxicities. Two other modified natural antitumor vinca alkaloids, vindesine (Eldesine®)8 3 and vinorelbine (Navelbine®)9 4 have been developed as antitumor compounds, and more recently, a fluorinated structural analog, vinflunine 5, was shown to possess potent antitumor properties10,11 and is now in phase III clinical trials in Europe (Figure 7.2). Thus, the discovery of vinblastine was a milestone in the development of cancer chemotherapy, inducing extensive chemical, biological, and clinical studies that finally led to the discovery of new potent antitumor vinca alkaloids. III. MECHANISM OF ACTION Soon after the discovery of the in vivo antitumor properties of the vinca alkaloids, a number of studies focused on their mode of action. Vinblastine was found to be a cell cycle–dependent OH H F F N N N N N N N N N H H H H3CO2C H H 3CO2C H H 3CO2C H H H 3CO OH 3CO OCOCH3 H CO OCOCH 3 3 N H N H HO CONH2 HO CO2CH N H 3 CO2CH3 CH3 HO CH3 CH 3 4 3 5 FIGURE 7.2 Vindesine (3), Vinorelbine (4), and Vinflunine (5). Copyright © 2005 CRC Press, LLC antimitotic agent that interacts with tubulin,12 a ubiquitous heterodimeric protein present in all eukaryotic cells. Tubulin and its polymerized form, microtubules, play crucial roles in the mainte- nance of cellular morphology and intracellular transport and in the construction of the mitotic spindle during cell division.13 The formation of microtubules is a dynamic process involving the assembly of tubulin and the disassembly of the polymers. The antiproliferative activity of the vinca alkaloids was shown to be a result of their interaction with the mitotic spindle.14 The alkaloids inhibit the assembly of tubulin into microtubules and, consequently, prevent the cells from under- going division. They bind to β−tubulin at different sites from colchicine and the antitumor taxoids, but a number of other natural products such as Rhizoxin, maytansine, cryptophycins, and dolastatins share the same binding site, interacting with the so-called vinca domain of tubulin.15–17 In vitro, the effects of vinca alkaloids on tubulin are concentration dependent. At low concentrations (sub- micromolar), they inhibit the formation and function of microtubules from tubulin, whereas spirals are formed at higher concentration.18,19 Depending on their structure, the effects of vinca alkaloids on microtubule dynamics are different, and these effects have been associated with the differences observed in the alkaloids’ efficacies and neurotoxicities. 20 Although the cellular target of the vinca alkaloids has been known since the 1970s, the precise location of the binding site is still unknown. Nevertheless, cross-linking experiments indicate that vinblastine interacts with the β-subunit of tubulin,21 and a recent synthesized fluorescent analog of vinblastine should give information about the binding site of vinblastine in the near future.22 IV. SEMISYNTHESIS AND TOTAL SYNTHESIS OF VINCA ALKALOIDS The availability of bioactive natural substances is often a major problem when they have to be produced on a commercial basis. Vinblastine 1 and vincristine 2 are isolated in very low yields from the leaves of C. roseus. Isolation yields of vincristine are on the order of 0.0003% from the dried leaves. Vinblastine is obtained in higher yield (0.01%) and has thus been used to produce vincristine, with oxidation of the indolinyl N-methyl group of the former leading to the latter. Vindesine 3 is also prepared from vinblastine by hydrazinolysis and subsequent hydrogenolysis of the newly formed N–N bond.8 Although cultivation of C. roseus provides a renewable source of vinblastine, a number of teams have studied the potential of preparing the antitumor vinca alkaloids by semisynthesis and total synthesis. The structural complexity of these molecules made this a significant synthetic challenge. The successful strategy of the semisynthesis of vinca alkaloids was based on a biogenetic hypothesis involving vindoline 7 and catharanthine 6 as precursors. These alkaloids are two of the more abundant alkaloids isolated from C. roseus, and the idea was that vinblastine 1, as well as other binary vinca alkaloids, could result from the union of vindoline 7 with an intermediate derived from catharanthine 6. After a number of attempts,1 a vinblastine-type alkaloid possessing the C18′S configuration, essential for bioactivity, could be obtained by applying a modification of the Polonovski reaction, also called the Polonovski–Potier reaction, to the Nb-oxide of catharanthine 8 and vindoline 7. Thus, treatment of catharanthine Nb-oxide 8 and vindoline 7 with trifluoroacetic anhydride led, after reduction by sodium borohydride, to the new bisindole alkaloid anhydrovin- blastine (9), which possesses the natural 18′S configuration (Scheme 7.1).23–25 Semisynthesis of anhydrovinblastine 9 was also realized from catharanthine and vindoline through ferric ion-mediated coupling in acidic aqueous media.26 Anhydrovinblastine was then found to be a natural vinca alkaloid, first by feeding experiments of both radioactive catharanthine and vindoline to C. roseus,27 and second by its extraction from the plant.28 More recently, a basic peroxidase with anhydrovinblastine synthase activity was purified from the leaves of C. roseus and was shown to produce the dimer from catharanthine and vindoline.29 Copyright © 2005 CRC Press, LLC N N O- H N N N+ H OCOCH 3CO 3 H CO2CH NH 3 CO2CH CH OH 3 7 N 3 N oxidation N H CO H 2CH3 H CO2CH H3CO OCOCH 3 1) Polonovski-Potier reaction 3 6 8 2) reduction N 9 H CO CH3 CH OH 2 3 SCHEME 7.1 Semisynthesis of anhydrovinblastine from catharathine. OAc + H N N 2/Pd N 1) m-CPBA Tl(OAc)2 NaBH4 9 1 N H 2) (CF3CO)2O N N V CO2CH H Polonovski V CO CH H CO 2 3 V 2CH 3 3 10 oxidation 11 V = vindoline SCHEME 7.2 Conversion of anhydrovinblastine to vinblastine. Because of its facile preparation from catharanthine and vindoline, anhydrovinblastine 9 was considered an attractive key intermediate in the synthesis of other binary alkaloids.1 Thus, vinblas- tine was obtained from 9 through different steps involving hydrogenation to 4′-deoxyleurosidine 10, Nb-oxidation and Polonovski reaction leading to enamine 11, treatment with thallium triacetate, and reduction with hydrolysis of the C4′ acetoxy group (Scheme 7.2).30 Another improved way to provide vinblastine from anhydrovinblastine was to subject enamine 11 to FeCl3-promoted oxygenation.31 Two other strategies have also been used to generate the C18′S–C15 bond of vinblastine 1 from natural vindoline 7 and substrates that could lead to the velbanamine part of the binary indole–indoline alkaloids.1 The first involved reaction of chloroin- dolenine 12 with silver tetrafluoroborate and vindoline 7 and led to intermediate 13, which produced vinblastine 1 after cyclization and deprotection (Scheme 7.3).32 In the second strategy (Scheme 7.4), the coupling methodology is based on a nonoxidative cleavage of the tertiary amine 14 that undergoes fragmentation after treatment with ClCO2CH2C6H4NO2 and vindoline 7.33 The indole–indoline dimer 15 was then converted to hydroxy aldehyde 16, which led to vinblastine 1 after hydrogenolysis and reduction of the iminium species 17. A new total synthesis of vinblastine was achieved recently by coupling chloroindolenine 19 with vindoline 7 synthesized from 7-mesyloxyquinoline 18 (Scheme 7.5).34 The iminium salt intermediate was formed by activation of 19 by trifluoroacetic acid, and electrophilic substitution with vindoline 7 led to dimer 20, which was cyclized to vinblastine after deprotection of the tertiary alcohol and amine groups. If anhydrovinblastine 9 is an important intermediate in the semisynthesis of a number of natural binary alkaloids, it is also the precursor of two unnatural anticancer drugs of the Vinca family: vinorelbine 4 and vinflunine 5. The first synthesis of vinorelbine came from the application of the Ph Ph + Ph N OTs N OTs OTs 1) Vindoline 7 (V) N OTMS AgBF 2) NaBH4 OTMS 4 OTMS 1 N N N H CO H CO Cl CO 2CH3 2CH 3 2C 3 V 12 13 SCHEME 7.3 Conversion of chloroindolenine (12) to vinblastine. Copyright © 2005 CRC Press, LLC CO2CH2C6H4NO2 O ClCO2CH N 2C6H4NO2-p vindoline 7= V O N N N H CH3NO2, -20C H CO2CH3 MeO2C O V O 14 1) H30 + 15 2) Py/SO3 CO OH 2CH2C6H4NO2 + CHO N N H /Pd/C 1 2 OH N N H CO2CH H 3 CO2CH3 V V 17 16 SCHEME 7.4 Conversion of tertiary amine (14) to vinblastine. MsO N 18 Ns OCOCF3 N Ns Ns N Cl (-)-vindoline 7 = V OCOCF3 N OCOCF3 1 TFA, CH2Cl2 + OTs 0C -> rt N N N CO2Me CO CH OTs H H 2 3 H3CO2C V OTs 19 20 SCHEME 7.5 Conversion of chloroindolenine (19) to vinblastine. Polonovski–Potier reaction to anhydrovinblastine Nb-oxide 21 (Scheme 7.6). Treatment of 21 with trifluoroacetic anhydride led to an unstable quaternary ammonium salt 22, which undergoes frag- mentation to a bisiminium salt 23. Addition of water and subsequent loss of formaldehyde produces a nucleophilic secondary amine 24 that gives rise to vinorelbine 4 after intramolecular trapping of the iminium group.9 Improvement in the synthesis of vinorelbine 4 could be obtained by using the chloro- or bromo-indolenines of anhydrovinblastine 25.35,36 OCO2CF3 O- + + N N N N N N H CO C H V 2 H3 V CO2CH H 3 V CO2CH3 21 9 (V = vindoline 7) 22 + N HN N + + N N N H V CO2CH3 H V CO2CH3 H V CO2CH3 4 24 23 SCHEME 7.6 Conversion of anhydrovinblastine Nb-oxide (21) to vinorelbine (4). Copyright © 2005 CRC Press, LLC V. MEDICINAL CHEMISTRY The medicinal chemistry of the vinca alkaloids has been extensively reviewed by Pearce37 and Borman and Kuehne.38 Since the discovery of their antitumor properties, many derivatives have been synthesized in the pharmaceutical industry, with the aim of improving their pharmacological activities to identify new drugs exhibiting a wider spectrum of clinical efficacy. The natural vinblastine 1 extracted from C. roseus leaves was used as a starting material because for a long time it was the only compound available in sufficient quantities. Briefly, most of the new analogs have been obtained by modifications of the vindoline “lower” part, bearing several reactive functions. Many such derivatives, including vinglycinate 26, vindesine 3, and vinzolidine 27, have been synthesized and evaluated by the Eli Lilly group (Figure 7.3). Vingly- cinate 26, with a glycine residue at the vindoline C4 position, was the first vinblastine analog to enter phase I clinical trials in 1967.39 Subsequently, modifications at the C3 position led to the amido- derivative vindesine 3,8 which can be considered a chemical precursor of vinzolidine 2737 and vintriptol 28.40 Vinepidine 29, corresponding to 4′-epi-4′-deoxyvincristine, was also developed by the Lilly group for its increased tubulin affinity relative to vinblastine 1.37 Nevertheless, none of these semisynthetic compounds showed marked benefits in clinical evaluations relative to vinblastine 1 and vincristine 2. In the meantime, as previously mentioned, the preparation of anhydrovinblastine 9 from catha- ranthine 8 and vindoline 7 facilitated the access to vinca alkaloids analogs exhibiting antimitotic properties,24 leading to vinorelbine 4 and vinflunine 5. To date, the semisynthetic compounds that have been approved for clinical use as anticancer drugs are vindesine 3 and vinorelbine 4. Although most of the work reported has been on the synthesis of analogs modified in the “lower half,” two reviews have appeared recently describing the consequences of modifications in the velbanamine “upper part” of the vinblastine skeleton.41,42 After 1990, only a few new derivatives have been prepared and evaluated with the aim of discovering novel anticancer drugs with clinical efficacy. These include the aminophosphonate derivative vinfosiltine 30 (Figure 7.3), selected for its unusual high potency both in vitro and in vivo compared to the classical vinblastine and vincristine.43 Vinfosiltine was designed on the basis of the similarity between α-amino phosphonic acids and natural amino acids. However, no evidence of marked benefice in phase II clinical trials, relative to other vinca alkaloids, has

been obtained, and development of vinfosiltine was discontinued in 1995.44 The natural anhydrovinblastine 9, biogenetic precursor of the dimeric vinca alkaloid, entered phase I clinical trials in 1999 and is claimed to be under phase II investigation in the United States as an agent against non–small cell lung cancer (NSCLC).45 OH OH OH N N N N N N N N N H H H H O 3CO2C H H 3CO2C H3CO2C H H H3CO O N H3CO OCOCH3 H3CO OH N H HO CO2CH N H O N H O 3 CH CH O HO 3 3 CH N 3 HN 26 27 O Cl 28 EtOOC OH N N H N H N N N N H H H3CO2C H3CO2C H H H3CO OCOCH3 H3CO OH N H HO CO2CH N H O 3 HO O O H CH3 OEt HN P 29 30 OEt FIGURE 7.3 Vinglycinate (26), Vinzolidine (27), Vintriptol (28), Vinepidine (29), and Vinfosiltine (30). Copyright © 2005 CRC Press, LLC OH N H O OH HO O N N 31a R = O O O H 31b R = H H H N N H N N 3CO2C N H N N H H H H 3CO OR O O O N H HO HO CO2CH3 OH CH3 H2N O FIGURE 7.4 Vinblastine-peptide derivative (31a) and desacetylvinblastine (31b). In a different domain, targeted delivery of vinca alkaloids has also been investigated. Several experiments were conducted during the 1980s with conjugates of vinblastine with monoclonal antibodies to deliver the cytotoxic drug to malignant tissues, but no further clinical development has been reported.46 More recently, a peptide–vinblastine derivative 31a targeted at prostate cancer cells has been reported (Figure 7.4).47 The proteolytic activity of prostate-specific antigen has been exploited to convert the conjugate 31a to deacetyl-vinblastine 31b at the site of the tumor, as demonstrated by in vivo studies using prostate-specific-antigen-secreting implanted human prostate cancer cells on nude mice. It should be pointed out that the putative effective drug 4-deacetylvin- blastine 31b, resulting from the hydrolysis of 31a, exhibited no in vivo activity under the same experimental conditions. Kuehne’s group has extensively documented the synthesis of vinblastine derivatives, mainly modified at the piperidine ring D′ of the velbenamine part.32,48 As a result it was clearly shown that subtle modifications at the C4′ position dramatically affect the interaction with tubulin49 together with cytotoxicity.50 More recently, two series of homo-vinblastine derivatives 32 and 33 (Figure 7.5) have been synthesized by the same group, highlighting the possible isolation of atropoisomers exhibiting in vitro activities different from those of the compound bearing the natural conformation. The authors also mentioned the concept of “thermal pro-drug activation” potentially applicable to these compounds.51,52 Among them, the 7′a-homo-vinblastine 32, including a 10-membered ring C′, was able to inhibit the tubulin polymerization at submicromolar concentrations, similar to vinblastine, but was slightly less cytotoxic than vinblastine when tested against the murine L1210 and S180 cell lines. However, the 18′a-homo-derivatives were shown to be inactive under the same experimental conditions. In a more recent publication, a large series of 62 vinblastine congeners was prepared and evaluated for their in vitro pharmacological properties.53 As an example, derivative 34, including an additional cyclohexane ring fused at C3′–4′ (Figure 7.6) induced a very high level of cytotoxicity toward the L1210 murine leukemia cell line (IC50 < 1 pM compared with 0.4 nM for vinblastine). However, the activity of 34 with RCC-2 cells (rat colon cancer) was slightly lower than that of the parent compound. In a similar way, it was shown that further ring D′ modified compounds such as 35 and 36 were as potent as vinblastine in terms of cytotoxicity (Figure 7.6).53,54 Surprisingly, superacid chemistry applied to vinca alkaloids resulted in access to newly modified compounds, specifically at the C4′ and C20′ positions, including vinflunine 5.10 These include 7' N N HO HO H H 18' N N N N H H H3CO2C H3CO2C H H H3CO OCOCH3 H3CO OCOCH3 N H HO CO2CH N H 3 HO CO2CH3 CH3 CH3 32 33 FIGURE 7.5 Homo-vinblastine derivatives. Copyright © 2005 CRC Press, LLC H N N N H N N N N N N H H H H3CO2C H C H3CO2C H 3CO2 H H H3CO OCOCH3 H3CO OCOCH3 H3CO OCOCH3 N H N H C HO CO N 2CH H 3 CO2CH H CH HO 3 3 3 CH HO CO2CH3 3 34 35 36 FIGURE 7.6 Ring D′ modified analogs. compounds 37a and 37b, substituted by halogen atoms (fluorine or chlorine) when the reaction was conducted in the presence of chlorinated solvents, and oxygenated derivatives 37c–e or 38 (alcohols or ketones) in the presence of hydrogen peroxide (Figure 7.7).41 A series of such derivatives has been evaluated for their overall pharmacological properties: tubulin polymerization inhibition, cytotoxicity, and in vivo antitumor activity against the P388 murine leukemia model. However, a lack of correlation between in vitro and in vivo results was revealed, and no clear structure-activity relationships were obtained. In the tubulin polymerization inhibition assay, all the compounds of the study exerted an activity in the micromolar range of IC50 close to those of the reference compounds. Clearly, this assay was not discriminatory enough to be useful in the selection of such derivatives. More surprisingly, binding of [3H]-vinflunine to tubulin was undetectable using the standard centrifugal gel filtration, unlike the other vinca alka- loids.55 This observation could appear somewhat paradoxical considering the overall in vivo anti- tumor activity of vinflunine 5. However, cytotoxicity values against the L1210 murine leukemia cells correlated with the induction of tubulin spirals for a subset of these compounds.56 Detailed investigations of pharmacokinetic and metabolism properties did not provide adequate results to understand this unusual profile of activities.57 It should be pointed out that alcohol 38, isolated after reaction in superacidic medium in 85% yield, corresponds to a minor metabolite of vinorelbine 4.58 Addition of fluorine atoms in vinorelbine 4 (resulting in vinflunine 5, for example) could block a metabolic pathway and modify its phar- macodynamic properties. The data available, however, do not explain the unusual pharmacological profile of 5: Similar to the other vinca alkaloids, vinorelbine is poorly metabolized, and compound 38, found at very low levels, is not a circulatory metabolite.59 Should superacids be considered as tools for biomimetic reactions under particular conditions? Or, on the contrary, should certain cytochromes be able to catalyze superacidic reactions? Further experiments need to be undertaken to validate this hypothesis. Taken together, all the results available in the literature appear to indicate that the strength of interaction with tubulin is not a sufficient criterion for the selection of a vinblastine derivative for further pharmacological studies. A quantitative comparison permitted the establishment of relative binding affinities for tubulin of vincristine > vinblastine > vinorelbine, in parallel with the ability to induce the formation of tubulin spirals, which are considered to be responsible for the neuro- toxicity observed as an undesirable side-effect in the clinic.60 R R 1 2 (CH C-4' 2) n R1 R2 R n 3 OH R3 N 4' N 37a 1,2 H H Cl R N N 37b 1,2 F H Cl R and S N N H H H3CO2C 37c 1 H ---O--- R H3CO2C H H H3CO OCOCH3 37d 1 F H OH R H3CO OCOCH3 N H N H HO CO2CH3 HO CO 1 2CH CH 37e OH H F S 3 3 CH3 37 38 FIGURE 7.7 Superacid-generated analogs. Copyright © 2005 CRC Press, LLC The cytotoxic activity is also not sufficient on its own: a very potent compound such as vinfosiltine 30 may demonstrate a high in vivo antitumor activity, but within a sharp dose range, resulting in a low therapeutic index. On the contrary, vinflunine 5 exhibited markedly lower cytotoxic properties in a panel of several murine and human cell lines compared to the standard vinca alkaloids55 but was the most active compound in a series of in vivo experiments that included murine models61 and human xenograft models.11 Microtubules are dynamic structures that are continuously shortening and growing. Detailed investigations evaluating microtubule dynamics perturbations have demonstrated that vinorelbine 4 and vinflunine 5 have a qualitative mode of action different from vinblastine 1.20 Moreover, examinations of the intracellular concentrations of vinorelbine 4 and vinflunine 5 compared with vinblastine 1 indicated the possible presence of sequestered drugs in “intracellular reservoirs.”62 The effects of vinca alkaloids on centromere dynamics have also been studied, leading to the conclusion that suppression of microtubule dynamics is the primary mechanism of action by which vinca alkaloids block cell mitosis.63 Overall, all these observations may contribute, in part, to the original in vivo efficacy of these new derivatives, but a correlation between tubulin interactions and antitumor activity remains to be established. Nevertheless, based on the numerous results obtained in the studies mentioned above, it appears clear that chemical modifications in the piperidine ring D′ or at the C4′ position induce dramatic changes in the pharmacological properties of vinca alkaloids. Despite the recent determination of the structure of the α,β-tubulin dimer resolved by electron crystallography64 and the subsequent studies aimed at improving the crystal resolution, the so-called vinca binding site remains insuffi- ciently known to investigate drug–protein interactions at the molecular level, rendering the rational design of more selective compounds almost impossible. To date, only a hypothetical model in which vinblastine 1 binds at the “plus end” interface of the polar part of the β-tubulin subunit has been proposed.65,66 Further investigations involving new crystallographic techniques, for example, will certainly allow a better understanding of the mechanism of action of vinca alkaloids in a near future. VI. CLINICAL APPLICATIONS The vinca alkaloids have been used in both curative and palliative chemotherapy regimens in clinical oncology for approximately 40 yr. A comprehensive review of their clinical applications can be found in Cancer: Principles and Practice of Oncology.67 The recent availability of vinorelbine has resulted in renewed interest in this class of compounds.68,69 Vincristine 1 plays a major role in combination chemotherapy in the treatment of acute lym- phoblastic leukemias and lymphomas. Vinblastine 2 is commonly used in combination with other anticancer drugs to treat bladder and breast cancers and is an essential component in the curative regimen for Hodgkin’s disease. Vinorelbine 4 has been approved worldwide for treating NSCLC either as a single agent or in combination with cisplatin. It has also been registered for advanced breast cancer in Europe, and an oral formulation is now available. Vinflunine 5 entered phase III clinical evaluations in 2003 in Europe against NSCLC and bladder cancer.70 Neurotoxic effects are the main side-effects observed with vincristine 1 and, to a lesser extent, with the other vinca alkaloids. These toxic effects have been associated with the affinity for axonal microtubules71 and with the ability to induce microtubule spirals, as mentioned before.56 Neutro- penia is the principal dose-limiting toxicity of vinca alkaloids, but recovery occurs after treatment. The emergence of drug-resistant cells is the major limitation on the clinical usefulness of vinca alkaloids, as for many other anticancer drugs.72 The best described mechanism involves the ampli- fication of the multidrug resistance protein, resulting in an efflux of the drug out of the cells mediated by the phosphoglycoprotein (Pgp) pump. However, recent studies with vinflunine demonstrated that the level of cross-resistance is much lower than that observed with vinorelbine or vincristine, Copyright © 2005 CRC Press, LLC which has positive implications for its clinical usage.73 Resistance phenomena have been extensively studied, and numerous reviews covering these aspects appear regularly in the literature.74,75 The discovery of the antiangiogenic properties of vinca alkaloids in the early 1990s offered new research areas with potential clinical applications.72,76 Moreover, recent studies highlighted the definite antiangiogenic activity of vinblastine at noncytotoxic doses,77 as demonstrated both in vitro on endothelial cells and in vivo using the chick embryo chorioallantoic assay. In addition, newer data indicate that vinflunine mediates its antitumor activity at least in part via an antivascular mechanism.78 These observations will certainly lead to new clinical applications of the vinca alkaloids when used at subtherapeutic doses, such as potentiation of standard cytotoxic compounds or potentiation of specific antiangiogenic derivatives. The association of taxanes with vinca alkaloids such as vinorelbine has revealed evidence of potential synergy in preclinical models,79 supporting a promising clinical efficacy, particularly in breast cancer. Finally, “new” targets of

vinca alkaloids may be discovered, and their participation in the antitumor and anticancer activities will have to be established. As an example, vinorelbine has been described as a potent calmodulin binder, inhibiting the association of calmodulin to its own target proteins.80 Further research is underway to investigate the potential role of calmodulin inhibitors such as vinca alkaloids and other tubulin interacting agents in the cellular division.81 VII. CONCLUSIONS Although the anticancer activity linked to clinical efficacy of vinca alkaloids has been recognized for more than three decades, further studies in different domains remain to be undertaken to better understand their mechanism of action. Recent advances in cell biology have identified numerous regulators of cell cycle such as molecular motors or specific kinases involved in the mitotic process. The question might be asked whether a mitotic blocker such as a new-generation vinca alkaloid could target certain of these proteins. Progress in structural biology should permit the determination of the tubulin structure at high resolution, allowing a better investigation of the interactions with vinca alkaloids at the molecular level. Then design of more selective molecules will be accessible with the aim of establishing the importance of the strength of interactions with tubulin for the antimitotic activity. In conclusion, continued research of novel tubulin interacting agents, including vinca alkaloids, might lead to more specific and more effective anticancer drugs. REFERENCES 1. Brossi, A. and Suffness, M., Eds. The Alkaloids, Vol. 37: Antitumor Bisindole Alkaloids from Catha- ranthus roseus (L.). Academic Press, Inc., San Diego, 1990. 2. Svoboda, G.H. and Blake, D.A., The phytochemistry and pharmacology of Catharanthus roseus (L.) G. Don, in Catharanthus Alkaloids, Taylor, W.I. and Farnsworth, N.R., Eds, Marcel Dekker, Inc., New York, 1975, chap. 2. 3. Noble, R.L., Beer, C.T., and Cutts, J.H., Role of chance observation in chemotherapy: Vinca rosea, Ann. N. Y. Acad. Sci., 76, 882, 1958. 4. Johnson, I.S., Wright, H.F., and Svoboda, G.H., Experimental basis for clinical evaluation of anti- tumor principles from Vinca rosea Linn, J. Lab. Clin. Med., 54, 830, 1959. 5. Svoboda, G.H., Alkaloids of Vinca rosea Linn. (Catharanthus roseus). 1X: Extraction and character- ization of leurosidine and leurocristine, Llyodia, 24, 173, 1961. 6. Neuss, N. et al., Vinca alkaloids, XXI. The structure of the oncolytic alkaloids vinblastine (VLB) and vincristine (VCR), J. Am. Chem. Soc., 86, 1440, 1964. Copyright © 2005 CRC Press, LLC 7. Moncrief, J.W. and Lipscomb, W.N., Structure of leurocristine methiodide dihydrate by anomalous scattering methods: relation to leurocristine (vincristine) and vincaleukoblastine (vinblastine), Acta Cryst., 21, 322, 1966. 8. Barnett, C.J. et al., Structure-activity relationships of dimeric Catharanthus alkaloids. 1. Deacetylvin- blastine amide (vindesine) sulfate, J. Med. Chem., 21, 88, 1978. 9. Mangeney, P. et al., 5′-nor anhydrovinblastine, prototype of a new class of vinblastine derivatives, Tetrahedron, 35, 2175, 1979. 10. Fahy, J. et al., Vinca alkaloids in superacidic media: a method for creating a new family of antitumor derivatives, J. Am. Chem. Soc., 119, 8576, 1997. 11. Hill, B. et al., Superior in vivo experimental antitumor activity of vinflunine, relative to vinorelbine, in a panel of human tumor xenografts, Eur. J. Cancer, 35, 512, 1999. 12. Lee, J.C., Harrison, D., and Timasheff, S.N., Interaction of vinblastine with calf brain microtubule protein, J. Biol. Chem, 24, 9276, 1975. 13. Dustin P., Microtubules, 2nd ed., Springer-Verlag, Berlin, 1984. 14. Jordan, M.A., Thrower, D., and Wilson, L., Mechanism of inhibition of cell proliferation by Vinca alkaloids, Cancer Res., 51, 2212, 1991. 15. Hamel, E., Natural products which interact with tubulin in the Vinca domain: maytansine, Rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B, Pharm. Ther., 55, 31, 1992. 16. Shih, C. and Teicher, B.A., Cryptophycins: a novel class of potent antimitotic antitumor depsipeptides, Curr. Pharm. Design, 7, 1259, 2001. 17. Gupta, S. and Bhattacharyya, B., Antimicrotubular drugs binding to the Vinca domain of tubulin, Mol. Cell. Biochem., 253, 41, 2003. 18. Himes, R.H. et al., Action of the Vinca alkaloids, vincristine, vinblastine and desacetyl vinblastine amide on microtubules in vitro, Cancer Res., 36, 3798, 1976. 19. Zavala, F., Guénard, D., and Potier, P., Interaction of vinblastine analogues with tubulin, Experientia, 34, 1497, 1978. 20. Ngan, V.K. et al., Novel actions of the antitumor drugs vinflunine and vinorelbine on microtubules, Cancer Res., 60, 5045, 2000. 21. Rai, S.S. and Wolff, J., Localization of the vinblastine-binding site on beta-tubulin, J. Biol. Chem., 271, 14707, 1996. 22. Chatterjee, S.K. et al., Interaction of tubulin with a new fluorescent analogue of vinblastine, Biochem- istry, 41, 14010, 2002. 23. Potier, P. et al., Partial synthesis of vinblastine-type alkaloids, J. Chem. Soc., Chem. Commun., 670, 1975. 24. Langlois, N. et al., Application of a modification of the Polonovski reaction to the synthesis of vinblastine-type alkaloids, J. Am. Chem. Soc., 98, 7017, 1976. 25. Kutney, J.-P. et al., Studies on the synthesis of bisindole alkaloids II. The synthesis of 3′-4′-dehy- drovinblastine, 4′-deoxovinblastine and related analogues. The biogenetic approach, Heterocycles, 3, 639, 1975. 26. Vucovic, J. et al., Production of 3′,4′-anhydrovinblastine: a unique chemical synthesis, Tetrahedron, 44, 325, 1988. 27. Scott, A.I., Guéritte, F., and Lee, S.L., Role of anhydrovinblastine in the biosynthesis of the antitumor dimeric indole alkaloids, J. Am. Chem. Soc., 100, 6253, 1978. 28. Goodbody, A.E. et al., Extraction of 3′,4′-anhydrovinblastine from Catharanthus roseus, Phytochem- istry, 27, 1713, 1988. 29. Sottomayor, M. et al., Purification and characterization of α-3′,4′-anhydrovinblastine synthase (per- oxidase-like) from Catharanthus roseus (L.) G. Don., FEBS Lett, 428, 299, 1998. 30. Mangeney, P. et al., Preparation of vinblastine, vincristine and leurosidine antitumour alkaloids, J. Am. Chem. Soc., 101, 2243, 1979. 31. Kutney, J.-P. et al., A highly efficient and commercially important synthesis of the antitumor Catha- ranthus alkaloids vinblastine and leurosidine from catharanthine and vindoline, Heterocycles, 27, 1845, 1988. 32. Kuehne, M.E., Matson, P.A., and Bornmann, W.G., Enantioselective syntheses of vinblastine, leuro- sidine, vincovaline, and 20′-epi-vincovaline, J. Org. Chem., 56, 513, 1991. Copyright © 2005 CRC Press, LLC 33. Magnus, P. et al., Nonoxidative coupling methodology for the synthesis of the antitumor bisindole alkaloid vinblastine and a lower-half analogue: solvent effect on the stereochemistry of the crucial C-15′/C-18′ bond, J. Am. Chem. Soc., 114, 10232, 1992. 34. Yokoshima, S. et al., Stereocontrolled total synthesis of (+)-vinblastine, J. Am. Chem. Soc., 124, 2137, 2002. 35. Andriamializoa, R.Z. et al., Composés antitumoraux du groupe de la vinblastine: nouvelle méthode de préparation, Tetrahedron, 36, 3053, 1980. 36. Guéritte, F. et al., Composés antitumoraux du groupe de la vinblastine: dérivés de la nor-5′ anhy- drovinblastine, Eur. J. Med. Chem., 18, 419, 1983. 37. Pearce, H.L., Medicinal chemistry of bisindole alkaloids from Catharanthus, in The Alkaloids, Vol. 37: Antitumor bisindole alkaloids from Catharanthus roseus (L.). Brossi, A. and Suffness, M., Eds. Academic Press, Inc., San Diego, CA, 1990, 145. 38. Borman, L.S. and Kuehne, M.E., Functional hot spot at the C-20′ position of vinblastine, in The Alkaloids, Vol. 37: Antitumor bisindole alkaloids from Catharanthus roseus (L.). Brossi, A. and Suffness, M., Eds. Academic Press, Inc., San Diego, CA, 1990, 133. 39. Armstrong, J.G. et al., Initial clinical experience with vinglycinate sulfate, a molecular modification of vinblastine, Cancer Res., 27, 221, 1967. 40. Rao, K.S.P.B. et al., Vinblastin-23-oyl amino acid derivatives: chemistry, physicochemical data, toxicity, and antitumor activities against P388 and L1210 leukemias, J. Med. Chem., 28, 1079, 1985. 41. Fahy, J., Modifications in the “upper” or velbenamine part of the Vinca alkaloids have major impli- cations for tubulin interacting activities, Curr. Pharm. Design, 7, 1181, 2001. 42. Duflos, A., Kruczynski, A. and Barret, J.-M., Novel aspect of natural and modified Vinca alkaloids, Curr. Med. Chem. Anti-Cancer Agents, 2, 55, 2002. 43. Lavielle, G. et al., New alpha-amino phosphonic acid derivatives of vinblastine: chemistry and antitumor activity, J. Med. Chem., 34, 1998, 1991. 44. Adenis, A. et al., Phase II study of a new Vinca alkaloid derivative, S12363, in advanced breast cancer, Cancer Chemother. Pharmacol., 35, 527, 1995. 45. Ramnath, N. et al., Phase I and pharmacokinetic study of anhydrovinblastine every 3 weeks in patients with refractory solid tumors, Cancer Chemother. Pharmacol., 51, 227, 2003. 46. Laguzza, B.C. et al., New antitumor monoclonal antibody-Vinca conjugate LY203725 and related compounds, J. Med. Chem., 32, 548, 1989. 47. Brady, S.F. et al., Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity, J. Med. Chem., 45, 4706, 2002. 48. Kuehne, M.E. et al., Three routes to the critical C16′-C14′ parf relative stereochemistry of vinblastine. Syntheses of 20′-desethyl-20′-deoxyvinblastine and 20′-desethyl-20′-deoxyvincovaline, J. Org. Chem., 52, 4340, 1987. 49. Borman, L.S. et al., Single site-modified congeners of vinblastine dissociate its various anti-microtu- bule actions, J. Biol. Chem., 263, 6945, 1988. 50. Borman, L.S. and Kuehne, M.E., Specific alterations in the biological activities of C-20′-modified vinblastine congeners, Biochem. Phamacol., 38, 715, 1989. 51. Kuehne, M.E. et al., Synthesis of 5′a-homo-vinblastine and congeners designed to establish structural determinants for isolation of atropoisomers, J. Org. Chem., 66, 5303, 2001. 52. Kuehne, M.E. et al., The synthesis of 16′a-homo-leurosidine and 16′a-homo-vinblastine. Generation of atropoisomers, J. Org. Chem., 66, 531 7, 2001. 53. Kuehne, M.E. et al., Synthesis and biological evaluation of vinblastine congeners, Org. Biomol. Chem., 1, 2120, 2003. 54. Parish, C.A. et al., Circular dichroism studies of bisindole Vinca alkaloids, Tetrahedron, 54, 15739, 1998. 55. Kruczynski, A. et al., Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid, Biochem. Pharmacol., 55, 635, 1998. 56. Lobert, S. et al., Vinca alkaloids-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line, Biochemistry, 39, 12053, 2000. 57. Bennouna, J. et al., Phase I and pharmacokinetic study of the new Vinca alkaloid vinflunine admin- istered as a 10-min infusion every 3 weeks in patients with advanced solid tumours, Ann. Onc., 14, 630, 2003. Copyright © 2005 CRC Press, LLC 58. Yamaguchi, K. et al., Identification of novel metabolites of vinorelbine in rat, Xenobiotica, 28, 281, 1998. 59. Van Heugen, J.C. et al., New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces, J. Chromatogr. A., 926, 11, 2001. 60. Lobert, S., Vulevic, B., and Correia, J.J., Interaction of Vinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine, Biochemistry, 35, 6806, 1996. 61. Kruczynski, A. et al., Preclinical in vivo antitumor activity of vinflunine, a novel fluorinated Vinca alkaloid, Cancer Chemother. Pharmacol., 41, 437, 1998. 62. Ngan, V.K. et al., Mechanism of mitotic block and inhibition of cell proliferation by the semisynthetic Vinca alkaloids vinorelbine and its newer derivative vinflunine, Mol. Pharmacol., 60, 225, 2001. 63. Okouneva, T. et al., The effects of vinflunine, vinorelbine and vinblastine on centromere dynamics, Mol. Cancer Ther., 2, 427, 2003. 64. Nogales, E., Wolf, S.G. and Downing, K.H., Structure of the α,β-tubulin dimer by electron crystal- lography, Nature, 391, 199, 1998. 65. Downing, K.H. and Nogales, E., Crystallographic structure of tubulin: implications for dynamics and drug binding, Cell Struct. Funct., 24, 269, 1999. 66. Checchi, P.M. et al., Microtubule-interacting drugs for cancer treatment, Trends Pharm. Sci., 24, 361, 2003. 67. Rowinsky, E.K and Tolcher, A.W., Antimicrotubule agents, in Cancer: Principles and Practice of Oncology, 6th ed., DeVita, V.T., Hellman, S., and Rosenberg, S.A., Eds, Lippincott-Raven, Philadel- phia, PA, 2001, 431. 68. Johnson, S.A. et al., Vinorelbine: an overview, Cancer Treat. Rev., 22, 127, 1996. 69. Gregory, R.K. and Smith, I.E., Vinorelbine — a clinical review, Br. J. Cancer, 82, 1907, 2000. 70. Pierre Fabre Laboratories, Castres, France. Press release September 10, 2003; http://www.pierre- fabre.com. 71. Binet, S. et al., Immunofluorescence study of the action of navelbine, vincristine and vinblastine on mitotic and axonal microtubules, Int. J. Cancer, 46, 262, 1990. 72. Hill, S.A. et al., Vinca alkaloids: anti-vascular effects in murine tumour, Eur. J. Cancer, 29, 1320, 1993. 73. Etiévant, C. et al., Markedly diminished drug-resistance inducing properties of vinflunine (20′,20′- difluoro-3′,4′-dihydrovinorelbine) relative to vinorelbine, identified in murine and human tumour cells in vivo and in vitro, with clinical implications, Cancer Chemother. Pharmacol., 48, 62, 2001. 74. Dumontet, C. and Sikic, B.I., Mechanisms of action and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death, J. Clin. Oncol., 17, 1061, 1999. 75. Kavallaris, M., Verrills, N.M., and Hill, B.T., Anticancer therapy with novel tubulin-interacting agents, Drug Resistance Updates, 4, 392, 2001. 76. Baguley, B.C. et al., Inhibition of growth of colon 38 adenocarcinoma by vinblastine and

colchicine. Evidence for a vascular mechanism, Eur. J. Cancer, 27, 482, 1991. 77. Vacca, A. et al., Antiangiogenesis is produced by non-toxic doses of vinblastine, Blood, 94, 4143, 1999. 78. Kruczynski, A. and Hill, B.T., Vinflunine, the latest Vinca alkaloid in clinical development. A review of its preclinical anticancer properties, Crit. Rev. Oncol. Hematol., 40, 159, 2001. 79. Aapro, M.S. et al., Developments in cytotoxic chemotherapy: advances in treatment utilising vinorel- bine, Crit. Rev. Oncol. Hematol., 40, 251, 2001. 80. Molnar, A. et al., Anti-calmodulin potency of indol alkaloids in in vitro systems, Eur. J. Pharm., 291, 73, 1995. 81. Moisoi, N. et al., Calmodulin-containing substructures of the centrosomal matrix released by micro- tubule perturbation, J. Cell Sci., 115, 2367, 2002. Copyright © 2005 CRC Press, LLC 8 The Bryostatins David J. Newman CONTENTS I. Introduction II. Biological Activities III. Chemical Syntheses of Bryostatins IV. Clinical Trials of Bryostatin V. Future Sources of Bryostatins A. Aquaculture B. Actual Source of the Bryostatins C. Chemical Analogs VI. Conclusions References I. INTRODUCTION The bryostatins (Figure 8.1) are a class of highly oxygenated macrolides originally isolated by the Pettit group under the early National Cancer Institute (NCI) program (1955–1982) designed to discover novel antitumor agents from natural sources. The initial discovery (of bryostatin 3, Figure 8.1) was indirectly reported in 1970.1 Subsequent developments leading to the report of the isolation and x- ray structure of bryostatin 1 (Figure 8.1) in 1982,2 and the multiyear program that culminated in the isolation and purification of (currently) 20 bryostatin structures, have been well documented by a variety of authors over the years.3–9 Structurally, all of the molecules possess a 20-membered macrolactone ring. Modifying the description by Hale et al.,9 all bryostatins possess a 20-membered macrolactone in which there are three remotely substituted pyran rings that are linked by a methylene bridge and an (E)-disubstituted alkene; all have geminal dimethyls at C8 and C18 and a four-carbon side chain (carbons 4–1) from the A ring to the lactone oxygen, with another four-carbon chain (carbons 24–27) on the other side of the lactone oxygen to the C ring. Most have an exocyclic methyl enoate in their B and C rings, though bryostatin 3, in particular, has a butenolide rather than the C-ring methyl enoate, and bryostatins 16 and 17 have glycals in place of the regular C19 and C20 hydroxyl moieties. In the reviews by Hale9 and Mutter,7 18 structures (bryostatins 1–18) are listed; the remaining two structures are now thought to be desoxy-bryostatin 4 and desoxy-bryostatin 5 isolated from Lisso- dendoryx isodictyalis.10 The levels of all bryostatins so far isolated from natural sources have always been at the minuscule level, with the highest yield being that of bryostatin 10, of which 15 mg was isolated from 1.5 kg of wet animal in a sample collected in the Gulf of Aomori.11 The actual source of these macrolides is debatable, and the evidence for microbial involvement is presented in a later section. Copyright © 2005 CRC Press, LLC FIGURE 8.1 Structures of the bryostatins. II. BIOLOGICAL ACTIVITIES Almost all of the data presented in the following section have been derived from studies with bryostatin 1. However, the basic biology of all of the bryostatins is fundamentally the same; they simply differ in potencies in given systems. Bryostatin 1 exhibited some extremely interesting biological activities from the beginning. Initially, it demonstrated quite variable activities in the then-current in vivo assays that were used at NCI, which were using the P388 and L1210 murine leukemia lines in normal mice. There was Copyright © 2005 CRC Press, LLC very significant variability in the results from batch to batch, but ultimately, using material isolated from Bugula neritina collected in the Gulf of California and from bulked fractions from other collection sites, Pettit et al. were able to publish the structure and some biological activities of bryostatin 1 in 1982.2 During the initial biological workup of bryostatin fractions by NCI it was realized that the response to P388, L1210, and KB cells mimicked the responses shown by extracts from plants of the families Euphorbiaceae and Thymelaceae. The possibility of isolating a phorbol- like structure from Bugula seemed remote, however, as there had never been reports of either phorbol or daphnane-type structures being isolated from marine organisms.3 Over the last 20 years, bryostatin 1 has demonstrated a very wide range of biological activities, including immune stimulation, differentiation of transformed cells, and enhancement of cytotoxicity of other agents. Initial experiments by Blumberg’s group at NCI and their collaborators in subse- quent years demonstrated that bryostatin’s anticancer activity was probably based on its interactions with, and subsequent modulation of, protein kinase C isozymes (PKCs) in cells.12,13 PKC kinases transfer the terminal phosphate group from adenosine triphosphate following binding of diacylg- lycerol and, frequently, phosphatidyl serine to the PKC isozyme. As a result of these interactions, PKCs are frequently concentrated at the cytosolic surfaces of cell membranes and are active at low physiological concentrations of calcium ions, thus modulating the inositol triphosphate (IP3) cascade within the cell. PKCs can be classified into three major groupings: conventional (α,βI/βIII,γ), where Ca2+ is required for activation; novel (δ,ε,θ,η/L), which are independent of Ca2+; and nontypical (ξ,λ/ι), where no binding by phorbols is known. The first two groupings have regulatory and catalytic domains, and the regulatory domain in particular has two cysteine-rich domains known as CRD1 and CRD2. Using binding-displacement studies it was shown that bryostatins, phorbol esters (PEs), and diacylglycerol (DAG) all compete for the same binding site or sites on PKC, and through suitable modeling studies, an early pharmacophoric model for DAG and PE was successfully applied to bryostatin binding parameters and to other PKC activators,14–16 which has subsequently led to the derivation of simpler analogs of the bryostatins by Wender’s group (see following). The main binding sites in the bryostatins included the C1, C19, and C26 oxygen atoms (for convenience, all subsequent comments on chemical analogs will use the bryostatin 1 numeration). This was proven by using chemically modified bryostatins and by following their binding affinities with PKC isoforms (see table 1 in Mutter and Wills for relative binding affinities7), but in spite of studies with these agents and other related compounds following cell line responses, no complete explanation as to why the biological responses between bryostatin and PE are so different has emerged to date. When the specific effects of bryostatin 1 on PKC in cell lines were studied in detail, it was found that although bryostatin down-regulates PKC, the expression of PKC was not directly affected. From both x-ray studies of phorbol 13-acetate bound to murine PKC-δ and the nuclear magnetic resonance solution structure of a PKC-α CRD2 construct, it was determined that the phorbol esters sit in a polar groove that exists at the tip of the CRD. In addition, the binding of such an “activator” does not appear to effect any significant conformational change in the binding domain. Thus, the “activator” sits over the inside polar surface of the groove, creating a continuous hydrophobic surface over approximately one-third of the complexed protein. This hydrophobicity increase of the PKC-δ-phorbol ester complex probably promotes its insertion into the plasma membrane, where it can then engage in signaling related to tumorigenesis,17 and it is likely that when bryostatin 1 binds to the CRDs of PKCs, similar changes occur, though specific differences may well occur with different PKCs. Thus, with PKC-δ, where there appears to be a “stabilizing effect” that prevents insertion into the membrane and then its subsequent degradation via the ubiquitin–proteosome pathway, it is possible that the conformational changes are different than those that occur with PKC-α and PKC-δ, where they undergo down-regulation via the degradation pathway. For further details as to the potential route of degradation of ubiquitinylated PKCs, the reader should consult Scheme 2 in the Copyright © 2005 CRC Press, LLC review by Hale et al.9 Further evidence for specific binding site/overlaps is given in the recent publication from Hale’s group, in which they demonstrated, using solution nuclear magnetic resonance techniques that bryostatin 1 (or, more precisely, an analog with specific structural features), does overlap its binding sites with those of phorbol 13-acetate and phorbol-12,13- dibutyrate in the CRD2 site of human PKC-α.18 A recent review of PKC–drug interactions as potential antitumor therapies should be consulted for further information on PKCs themselves and on other compounds that may well function via these pathways.19 In addition to data on the interaction with PKCs, a very significant amount of evidence has accumulated that suggests that bryostatin 1 and, by inference, other similar compounds, but perhaps not the 20-deoxy class such as bryostatin 13, can function as very potent immunostimulants. Thus, resting T cells and neutrophils are activated both in vitro and in vivo,20–23 and in clinical trials, it has been shown to raise circulating levels of tumor necrosis factor-α (TNF-α) which is normally produced by the body following immunostimulation.24 In in vitro studies with the murine mac- rophage line, ANA-1, treatment with bryostatin 1 significantly increased TNF-α mRNA expression and also exhibited synergy with interferon-γ in the production of nitrite and the subsequent expres- sion of the inducible nitric oxide synthetase gene. This gene catalyzes the in vivo production of NO from L-arginine, and NO is known to produce strong tumoricidal effects on murine macroph- ages, probably via induction of the apoptotic cascade.25 Thus, bryostatins may well be exerting some of their effects via immunomodulation pathways. In contrast, bryostatin 13, a 20-deoxy-bryostatin, does not stimulate colony formation in bone marrow progenitor cells, whereas bryostatins 1, 3, 8, and 9 do. In addition, bryostatin 13 is claimed to be more potent as an antitumor agent than the other four.25 Thus, there may well be a component of immunostimulation in the antitumor activities of the C20-O-acyl bryostatins that is not present in the 20-deoxy class. The recent report by Battle and Frank has demonstrated that one potential mechanism of bryostatin 1–mediated differentiation in human CLL cells is activation of the signal transducer and activator of transcription (STAT). Thus, in cells taken from clinical trials patients, bryostatin 1 appears to activate STAT1 in a PKC-dependent manner by induction of an IFN-γ autocrine loop; this leads to activation of the JAK-STAT1 signaling pathway and to the ultimate differentiation of the cells.26 There are also reports that imply that bryostatin 1, under certain conditions, might have some tumor-inducing capability, though this is an extrapolation from cell line studies. In studies in which it was demonstrated that bryostatin 1 can selectively target PKC-II isozymes in K562 (human erythroleukemia) and HL60 (human promyelocytic) cell lines, such treatment led to membrane translocation and lamin B phosphorylation at specific sites on the lamin proteins. Such transloca- tion/phosphorylation causes breakdown of the nuclear envelope during mitosis, and in these cells, such an effect appears to be associated with enhanced proliferation.27 Thus, care might have to be taken in choosing patients for bryostatin treatment (see later section on clinical trials). III. CHEMICAL SYNTHESES OF BRYOSTATINS Since the publication of the first structure by Pettit in 1982, these molecules have been the target of many synthetic chemistry groups. Many partial syntheses have been published in which specific portions of the molecule have been made, but to date, only three of the bryostatins have been synthesized. The first was the enantioselective total synthesis of bryostatin 7 in 1990 by Masamune et al.,28 the second was by Evans et al. on the enantiomeric total synthesis of bryostatin 2 in 1999,29 and the third was the synthesis of bryostatin 3 by Nishiyama and Yamamura in 2000.30 In addition to these papers, three excellent review articles covering through 2002, on the syntheses of these three and other partial bryostatin structures including bryostatin 1, have been published and should be consulted for specific details of reaction schemes and comparisons of routes.7,9,31 Copyright © 2005 CRC Press, LLC IV. CLINICAL TRIALS OF BRYOSTATIN Although a number of bryostatins have been tested in animals and many have had in vitro assessments in many cell lines, only bryostatin 1 has entered human clinical trials. Part of the reason is that this is the material that could be obtained in quantities large enough to be able to isolate and produce the compound under cGMP conditions. The history of the initial attempts at large-scale isolations and