Clear cell carcinoma, morphologic features. A full color version of this figure is available at the Modern Pathology journal online. 

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... clear cell carcinoma is an uncommon histotype that has been under-represented in pro- spective therapeutic trials. 1–4 This tumor is less responsive to standard therapeutic regimens 4 and may be amenable to targeted therapeutic approaches. However, the molecular pathogenesis of clear cell carcinoma, on which any such approaches would be based, is unclear. 5 There have been two major analyses of the molecular features of clear cell carcinoma, and both groups independently concluded that this carcinoma exhibits molecular heterogeneity, a spectrum of molecular changes and/or may arise through multiple pathways. 6,7 Although the degree to which the aforementioned heterogeneity can be attributed to variations in the pathologic diagnosis of clear cell carcinoma 8 is unclear, these studies do suggest that there may be molecular subsets of the histotype that are worthy of evaluation for their possible clinicopathologic significance. The SWI/SNF (mating type switching/sucrose non-fermenting) complex is an evolutionarily con- served multiunit complex of factors that utilize the energy of ATP hydrolysis to remodel nucleosomes and thereby affect gene transcription. 9–13 Eukaryotic SWI/SNF complexes are comprised of two catalytic core subunits, and up to 10 non-catalytic subunits, one of which is BAF250a (the protein product of the ARID1A (adenine-thymine (AT)-rich interactive domain containing protein 1A) gene). 9,10 Inactivating somatic mutations of ARID1A have been identified in 46–57% of ovarian clear cell carcinomas, 14,15 and they appear to have a tumor suppressor role in this and some other gynecologic cancers. 14–16 Loss of BAF250a expression is not uncommon in endometrial cancers, 17–20 and has been reported in 22.7–26% of endometrial clear cell carcinomas. 18,20 In a previously reported pilot study of 22 clear cell carcinomas, loss of BAF250a expression was found to correlate with advanced stage at diagnosis on univariate analyses; 20 Comparable studies are conflicting on the question of its prognostic significance in ovarian clear cell carcinoma, 21–24 whereas recent studies suggest that loss of BAF250a may represent a negative prognostic factor in gastric and breast cancers. 25,26 The tumor suppressor gene TP53 (protein product p53) is the most commonly altered gene in human cancers. 27,28 Emerging lines of evidence indicate that the SWI/SNF complex and its subunits, including the ARID1A gene product BAF250a, are key regulators and targets of p53 function, and that ARID1A functions as a tumor suppressor by modulating the transcriptional activity of TP53 regulated genes. 16,29–35 In preliminary analyses, we have noticed that a small subset of BAF250a À clear cell carcinomas also show p53 overexpression, 5 but it was unclear if the coexistence of these phenotypes was entirely fortuitous. In this study, we systematically assess the clinicopathologic significance of p53 and BAF250a expression in a group of rigorously classified clear cell carcinomas of the endometrium, with the ultimate goal of determining if there are any singular or composite p53/BAF250a expression profiles that define biologically distinct subsets of this tumor. This study was approved by the institutional review board at Vanderbilt University (IRB no. 12606), and was based on an analysis of archived material from the authors’ institutions. The contributors, who are all gynecologic pathologists, searched their respec- tive files for cases diagnosed as endometrial clear cell carcinoma, reviewed the slides and retrieved cases whose morphologic features they considered to be unequivocally diagnostic of this histotype. A total of 62 cases were generated. These 62 cases were then reviewed by three authors (OF, VP and JH) independently. Each reviewer classified the 62 cases into two groups: endometrial clear cell carcinoma and a histotype other than clear cell carcinoma. Cases of mixed carcinoma with a clear cell component were classified as the latter. The sole prerequi- site for inclusion of a case into the final data set was that a diagnosis of clear cell carcinoma was rendered for that case by at least two of the three reviewers. Upon completion of the review, there was excellent diagnostic agreement between the three reviewers, with a k -value of 0.846. In 53 of 62 cases, the three reviewers had identical classifications (85% agreement rate). In all, 12 (19%) of the original 62 cases were ultimately classified as a histotype other than clear cell carcinoma after the review, leaving a final data set of 50 cases (Figure 1). Whole tissue sections from 5 biopsies and 45 hysterectomies were used for immunohistochemical analyses, which were performed on the Leica Bond Max immunohistochemical autostainer (Leica Mi- crosystems, Buffalo Grove, IL, USA). Heat-induced antigen retrieval was performed on the Bond Max using the Leica Epitope Retrieval 2 solution for 30 min (BAF250a) and 20 min (p53). For BAF250a, slides were incubated with the primary antibody (BAF250a, PSG3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 1:750 dilution. This antibody is a mouse monoclonal antibody that was raised against a recombinant protein that corre- sponds to amino acids 600–1018 of the human BAF250a protein. For p53, slides were incubated with the prediluted primary antibody (clone DO-7; Leica), a mouse anti-human monoclonal antibody. The Bond Polymer Refine detection system was used for visualization. This ready-to-use system is a biotin-free, polymeric horseradish peroxidase (HRP)-linked antibody conjugate system, and con- tains a peroxide block (to limit endogenous peroxidase activity), post-primary IgG linker reagent (to link mouse antibodies), poly-HRP reagent (to loca- lize rabbit antibodies), the substrate chromogen (3 0 ,3-diaminobenzidene tetrahydrochloride) and he- matoxylin counterstain. Slides were the dehydrated, cleared and coverslipped. Staining patterns were interpreted using different scoring methods (Figures 1b–f). Immunohistochemical staining for BAF250a was scored using a previously described system that incorporates both the extent and intensity of staining. 25 The extent was scored on a four-tiered semiquantitative scale based on the estimated percentage of tumor epithelial cells displaying any immunoreactivity: 0 (0–9%), 1 (10–25%), 2 (26–50%) and 3 (51–100%). Intensity of BAF250a immunoreactivity was similarly scored on a four-tiered scale (0–3). The final score for each case was obtained by multiplying the ‘extent’ score by the ‘intensity’ score, with potential final scores that thus ranged from 0 to 9. A final score of 0 was considered to be negative (Figure 1f), a score of 1–3 was considered weakly positive (Figure 1d) and a score of 4–9 was considered to be strongly positive (Figure 1e). BAF250a is a nuclear protein that is expressed to varying degrees in most human cells. As nuclear expression of BAF250a is an expected finding in lymphocytes, endothelial cells and stromal cells, 15,18 these cells served as internal positive controls for assay validity. P53 staining status was assessed based on previously described concepts on staining patterns that most likely correlate with an underlying TP53 mutation and/or have prognostic significance. 36–38 Cases were classified either as displaying a ‘p53-wild-type‘ pattern of staining (p53-wt: focal and/or weak and/or heterogeneous staining pattern; Figure 1c) or as ‘p53 þ ’ (strong, 3 þ , diffuse expression in at least half of tumoral nuclei (Figure 1b), or complete absence of staining in tumoral nuclei in the setting of wt staining of background non-epithelial cells: null phenotype). Kaplan–Meier survival curves were generated for overall survival and progression-free survival, defined by the period between primary treatment and death or relapse. Comparisons between survival curves were performed using log-rank tests. Cox regression analyses were used to assess relationships between clinicopathologic factors, including p53 and BAF250a expression, and outcome using multivariate and univariate models. Univariate analyses using Fisher’s exact, Pearson’s w 2 and Student’s t -tests were also used to compare between subgroups as appropriate. Spearman’s correlation tests were used to assess the relationships between p53 and BAF250a expression. A P -value of o 0.05 was considered to be statistically significant for all analyses. The 50 patients ranged in age from 50 to 85 (mean 67.8) years. Their International Federation of Gynecology and Obstetrics stage distribution was as follows: stage I ( n 1⁄4 19; IA, n 1⁄4 18, including one pT0; IB, n 1⁄4 1), stage II ( n 1⁄4 8), stage III ( n 1⁄4 14; IIIA, n 1⁄4 6; IIIB, n 1⁄4 1; IIIC, n 1⁄4 7)) and stage IV ( n 1⁄4 9). In total, 48 patients underwent a total hysterectomy with bilateral salpingo-oophorectomy, and 2 did not undergo a primary surgical procedure other than the diagnostic biopsy. Regional lymphadenectomy was performed in 43 patients, with only pelvic nodes removed in 10 patients, and both pelvic and paraaortic nodes removed in 23 patients. In 10 patients, lymph nodes were positive for metastatic disease. Among the 19 patients with stage I disease, lymphadenectomy was performed in 13 (pelvic only in 3; pelvic and paraaortic in 10). For the eight stage II patients, lymphadenectomy was performed in seven (pelvic only in two; pelvic and paraaortic in three). Overall, directed peritoneal biopsies and/or omentectomy was performed in only 16 of the 27 stage I or II patients. Adjuvant treatments included ...