{"paper_id":"5ce68f21-38a8-4737-986e-38e3543d663f","body_text":"ARID1A expression in ovarian clear cell carcinoma with an \nadenofibromatous component \n \n（腺線維腫を有する卵巣明細胞腺癌における Adenine-\nthymine–rich interactive domain 1A の発現について） \n \n \n \n \n千葉大学大学院医学薬学府 \n先端医学薬学専攻 \n(主任：生水真紀夫教授) \n錦見 恭子 \n \n \n \n \n  \n\nAbstract \nAims: The carcinogenesis of ovarian clear cell carcinoma (CCC) has been hypothesized to \ncomprise two different pathways: an adenofibroma-carcinoma sequence and an \nendometriosis-carcinoma sequence. However, the difference in the genetic basis of these two \npathways remains unclear. Recent studies have suggested that an ARID1A mutation and the \nloss of the corresponding protein, BAF250a, are frequent events in CCC. Herein, we \ninvestigated the difference in the loss of BAF250a expression in adenofibroma-related CCC \nand endometriosis-related CCC.  \nMethods and Results: In total, 93 cases of surgically treated CCC were evaluated. The \npresence of adenofibroma and endometriosis associated with carcinoma was determined by \nreviewing hematoxylin and eosin-stained slides for each case. BAF250a expression in \ncarcinoma was examined immunohistochemically. The loss of BAF250a expression was \ndetected in carcinomas in 50 of 93 (54%) cases, including 5/18 (28%) with adenofibroma \nalone, 30/45 (67%) with endometriosis alone, 8/18 (44%) with both conditions, and 7/12 \n(58%) with neither condition. The loss of BAF250a expression was significantly less frequent \nin CCC cases with adenofibroma than in cases with endometriosis (p = 0.01, Fisher’s exact \ntest). \nConclusions: The action of ARID1A in carcinogenesis differs between adenofibroma-related \nCCC and endometriosis-related CCC.  \n\nIntroduction \nAdenine-thymine–rich interactive domain 1A (ARID1A) has recently been identified as a \ntumour suppressor gene, which is associated with various human neoplastic lesions, including \ngynaecological cancers.1-5 Genome-wide sequencing analysis has shown approximately 50% \nof ovarian clear cell carcinomas (CCC) and 30% of ovarian endometrioid carcinomas, but \nnone in high-grade serous carcinoma, to harbour somatic mutations in ARID1A,2, 3 ARID1A \nencodes BAF250a, a component of the ATP-dependent chromatin remodelling complex \nSWI/SNF, containing BRG1 or BRM,2 which plays a crucial role in cell proliferation and \ndifferentiation.4-8 Recent studies in mice have shown that ARID1A (BAF250a) promotes the \ngrowth of ovarian carcinomas by interacting with p534, PTEN9, or PIK3CA.10 The loss of \nBAF250a expression—detected by immunostaining—has recently been shown to be \nsignificantly correlated with an ARID1A mutation,2,11 and it can therefore be used as a \nsurrogate marker.  \n Ovarian CCC is frequently associated with endometriosis and less often with \nadenofibroma. The carcinogenesis pathways of endometriosis-related CCC and \nadenofibroma-related CCC are hypothesized to differ from each other12-14 despite the fact that \nboth endometriosis and adenofibroma are occasionally present in association with the same \nCCC concurrently.15-17 The molecular abnormality underlying endometriosis-related CCC has \nbeen ascribed to the highly inflammatory, stressful environment of an endometriotic lesion, \n\nleading to the activation of the PTEN-PIK3CA-mTOR pathway.18 However, the molecular \nabnormality underlying adenofibroma-related CCC remains unclear. The present study was \nconducted to investigate differences in the loss of BAF250a expression in adenofibroma-\nrelated CCC and endometriosis-related CCC to clarify the difference in the genetic \nbackground and carcinogenesis between these two lesions.  \n \nMaterials and Methods \nPatients and tissue samples \nThe Institutional Review Board of Chiba University Graduate School of Medicine approved \nthis research protocol (Approval Number 1903, 21 August 2014). Ninety-three patients with \nCCC who were surgically treated between 2000 and 2012 at Chiba University Hospital and \naffiliated hospitals in Chiba, Japan, were included in the study. All available hematoxylin and \neosin-stained slides of the ovarian tumours were reviewed by two gynaecologic pathologists \nto confirm the histological type of the tumours on the basis of the WHO classification19 and \nfor the presence of adenofibroma and/or endometriosis associated with carcinoma. In the \npresent study, adenofibroma was defined by the presence of proliferation foci of tubules lined \nby a single layer of tumour cells, with minimal nuclear atypia and a marked stromal \nfibromatous component.15-17 We classified endometriosis into two categories: one with \nendometriotic cyst formation contiguous with or adjacent to CCC, and the other being distant \n\nfrom CCC, without endometriotic cyst formation. \n \nImmunohistochemical staining \nFormalin-fixed paraffin-embedded tissue sections (4-μm-thick) were deparaffinized, and \nimmunohistochemistry was performed using the antibody against ARID1A (BAF250a) \n(HPA005456, Sigma-Aldrich, Tokyo, Japan) at a dilution of 1:400. Auto-stainer Link48 \n(Dako, Tokyo Japan) was used for immunostaining. Antigen retrieval was performed by \nincubating sections in low pH Target Retrieval Solution (Dako) at 98°C for 20 min. \nImmunohistochemical staining was scored using a previously described method.1, 20 The \npercentage of positively stained epithelial cells was scored as 0 (0–9%), 1 (10–25%), 2 (26–\n50%), or 3 (51–100%), and the intensity was scored as 0 (undetectable), 1 (weak staining), 2 \n(moderate staining), or 3 (strong staining). The total immunostaining score was defined as \npercentage positivity score × staining intensity score and ranged from 0 to 9. We considered \ntotal immunostaining scores of 0–4 as loss of BAF250a expression and scores of 6 or 9 as \nretained BAF250a expression.  \n \nStatistical analysis  \nLoss of BAF250a expression in adenofibroma-related CCC was compared with that in \nendometriosis-related CCC using Fisher’s exact test. Logistic regression analysis was used to \n\nclarify the correlation between adenofibroma and loss of BAF250a expression after adjusting \nfor the presence of endometriosis. A survival curve for patients with pT1 was calculated using \nthe Kaplan–Meier method and compared by the log-rank test. Statistical analyses were \nperformed using the Statistical Package for Social Sciences, version II for Windows (SPSS \nInc., Chicago, IL, USA); p < 0.05 was considered statistically significant.  \n \nResults \nPatient characteristics and associated lesions  \nThe patients’ characteristics and BAF250a expression status are summarised in Table 1. All \npatients were surgically treated, with no residual tumour. The follow-up period ranged from \nseven months to 150 months, with a median of 60 months. Overall, 86 of 93 patients \npresented with stage pT1, and the remaining 7 presented with pT2 or pT3. Of 93 CCCs, 18 \n(19%) were associated with adenofibroma alone, 45 (48%) with endometriosis alone, 18 \n(19%) with both components, and 12 (13%) with neither of them. No patient with atypical \nendometriosis was identified. \n \nLoss of BAF250a expression in adenofibroma, endometriosis, and carcinoma \nNone of the 80 cases with adenofibroma and/or endometriosis showed a loss of BAF250a \nexpression in these components, and all had a total immunostaining score of 9. In contrast, \n\nthe extent of BAF250a immunoreactivity in the carcinomatous component varied with regard \nto staining intensity and the percentage of positive cells (Figure 1). Loss of BAF250a \nexpression (score 0–4) was detected in the carcinomatous component in 50 (54%) of 93 \npatients, and the breakup is as follows: 45 (52%) of 86 patients with pT1 disease, and 5 \n(71%) of 7 patients with pT2/pT3 disease. The difference between the two groups was not \nstatistically significant (p = 0.445, Fisher’s exact test). Patients with loss of BAF250a \nexpression in CCC included 5 (28%) of 18 with adenofibroma alone, 30 (67%) of 45 with \nendometriosis alone, 8 (44%) of 18 with both adenofibroma and endometriosis, and 7 of 12 \n(58%) with neither condition (Table 2). Loss of BAF250a expression was found to be \nsignificantly less frequent in CCC associated with adenofibroma alone than in CCC with \nendometriosis alone when analysed by Fisher’s exact test (p = 0.01). Because 18 patients had \nboth endometriosis and adenofibroma, data were adjusted for endometriosis to clarify the \ncorrelation between adenofibroma and loss of BAF250a expression (Table 3). Univariate and \nmultivariate analyses showed that the loss of BAF250a expression was less frequent in CCC \nwith an adenofibromatous component. In 63 CCC patients with endometriosis, loss of \nBAF250a expression in the carcinomatous component occurred in 26 (55%) of 47 patients \nwith endometriotic cysts contiguous with or adjacent to CCC and in 12 (75%) of 16 patients \nwith endometriosis distant from CCC without cyst formation. Loss of BAF250a expression in \nCCC did not differ between the two types of endometriosis (p = 0.24, Fisher’s exact test). \n\n \nPrognostic impact of BAF250a expression \nThe 5-year progression-free survival rates in the 86 patients with pT1 CCC with and without \nloss of BAF250a expression were 82% and 79%, respectively. Kaplan–Meier analysis \nrevealed no significant relationship between BAF250a immunoreactivity and progression-\nfree survival in pT1 CCC patients (p = 0.89, log-rank test) (Figure 2).  \n \nDiscussion \nIn this study, we have shown that loss of BAF250a expression was significantly less frequent \nin adenofibroma-associated CCC than in endometriosis-associated CCC. This is the first \nreport showing a genetic difference between adenofibroma-associated CCC and \nendometriosis-associated CCC. Our results suggest that the mutation in ARID1A, which \nencodes BAF250a, may not be as strongly associated with the development of adenofibroma-\nrelated CCC as it is with endometriosis-related CCC, and that the role of ARID1A in \ncarcinogenesis may differ between the two groups. We observed a loss of BAF250a \nexpression in only 28% in adenofibroma-related CCC cases, indicating that another non-\nARID1A genetic aberration might underlie this condition. In contrast, loss of BAF250a \nexpression in CCC with endometriosis was observed in 67% of the patients, consistent with \nprevious studies.2, 21, 23 This indicates that the ARID1A mutation plays an important role in the \n\ndevelopment of CCC derived from endometriosis. Furthermore, the high frequency of \nARID1A mutations in our study, as well as in earlier reports, and in ovarian endometrioid \nadenocarcinoma but not in high-grade serous carcinoma2, 3 suggests that ARID1A mutations \nmight be associated with carcinomas related to endometriosis. In evaluating BAF250a \nimmunostaining, we use the total score defined as percentage positivity score × staining \nintensity score ranging from 0 to 9.1, 20 Cells without ARID1A mutations, including normal \ncells, are expected to show positive staining for BAF250a. We considered ‘loss of BAF250a \nexpression’ as significant decreases in staining intensity and in percentage of stained cells, \nand used the cut off index as a score of 4 or less to avoid overestimating the loss of BAF250a \nexpression. We believe our evaluation method is justifiable, given that our result of BAF250a \nexpression in CCC with endometriosis was consistent with that of published studies.2, 21- 23 \nIn our study, loss of BAF250a expression was not observed in the adenofibromatous \ncomponent in any of the cases studied. This implies that ARID1A mutations do not occur in \nthese putative precursor lesions and are not early events in adenofibroma-related CCC. This \nis in conflict with the findings of Yamamoto et al., who reported the loss of BAF250a \nexpression in six of 14 cases of CCC associated with adenofibroma; benign (three of three), \nand borderline (six of six) clear cell adenofibroma components adjacent to carcinoma were \nfound to lack BAF250a expression.23 A possible reason for the discrepancy between our \nresults and those of Yamamoto et al. is the difference in the criteria used for adenofibroma \n\ndiagnosis. Diagnostic criteria for the distinction between clear cell adenofibroma and \nborderline adenofibroma, and between borderline adenofibroma and CCC, have not been \nfirmly established.15-17 Although we only designated cases as adenofibroma when tubules \nwith minimal nuclear atypia were present, we occasionally observed a few tubules with \nmoderate atypia mixed with tubules of carcinoma in the abundant fibrous stroma of \nadenofibroma-related CCC. In these cases, we considered the lesion to be part of the \ncarcinoma and thus did not include it as an adenofibroma component.  \nThere have been several reports of loss of BAF250a expression not only in carcinoma \nbut also in endometriosis and atypical endometriosis.2, 21-23 According to these reports, an \nARID1A mutation occurred in atypical endometriosis before the development of carcinoma, \nand it was an early event in the malignant transformation of ovarian endometriosis. In the \npresent study, we were not able to identify foci of atypical endometriosis in any of the cases \nincluded. Moreover, in contrast to some previous reports,21-23 the endometriotic component \ndid not show loss of BAF250a expression in our study, perhaps owing to differences in the \nhistological features of endometriotic lesions and experimental protocols. In our study, we \nincluded endometriosis associated with carcinoma, regardless of location and size. In most of \nour endometriosis patients, endometriotic lesions were subtle, often with a limited number of \nepithelial cells present. In studies by Ayhan et al. and Yamamoto et al., loss of BAF250a \nexpression was observed in the epithelial cells of endometriotic lesions but not in endometrial \n\nstromal cells.21, 23 Thus, it is possible that in our patients, the epithelial cells of some \nendometriotic lesions had indeed lost BAF250a expression, but this could not be detected \nbecause of epithelial exfoliation. Alternatively, methodological attributes for these \ndiscrepancies might include differences in the antibodies, antibody dilutions, and \nimmunohistochemical assessment methods used. To conclude whether ARID1A mutation \noccurs in conventional endometriosis and is an early event during malignant transformation \nof ovarian endometriosis, larger studies and additional methods such as DNA or RNA \nsequencing analysis2, 11 are needed.  \nThere are conflicting reports concerning the prognostic value of BAF250a expression in \nCCC. No prognostic impact of the loss of BAF250a expression was detected for pT1 CCC \ncases in our study. Maeda et al. reported no significant difference in overall survival in 121 \ncases of CCC with or without loss of BAF250a expression.11 In contrast, Katagiri et al. \nreported that CCC patients showing loss of BAF250a expression had a shorter progression-\nfree interval than those with normal levels of BAF250a.24 Both these studies included CCC \ncases of all stages (FIGO stages III/IV in 31 of 121 cases and 15 of 60 cases in the former and \nlatter studies, respectively), and that of Katagiri et al. included 12 of 60 cases with residual \ntumours of ≥2 cm, whereas in the present study, we included pT1 CCC cases only. It is \npossible that loss of BAF250a expression may be a prognostic factor in advanced-stage CCC; \nhowever, further in-depth studies are required to validate this concept.  \n\nIn conclusion, we identified a genetic difference in the carcinogenesis pathways of \nadenofibroma-related and endometriosis-related CCC by revealing that loss of BAF250a \nexpression was significantly less frequent in adenofibroma-related CCC than in \nendometriosis-related CCC. Our results suggest that other non-ARID1A genetic aberrations \nmight underlie adenofibroma-related CCC. We expect that our results might aid in the \ndiscovery of such mutations. Patients with CCC are resistant to currently available cytotoxic \ndrugs. Therefore, another implication of our present findings is the possibility of \nindividualization of CCC treatment once drugs targeting ARID1A are developed.  \n \n\nAcknowledgments \nWe thank Tsutomu Matsui, Kikuyo Kawashima, and Takashi Onodera for their technical \nassistance. We also thank Kouichiro Hirashiki, Youichi Unno, Yoshihiko Izawa, Naotake \nTanaka, Michio Sanada, Hideaki Iwasaki, Noriko Yamamoto, Hideo Matsui, Reiko \nMatsumoto, Yuka Sakuma, Nobuo Endo, Yoshimasa Kawarai, Masaaki Kamiyama, Takako \nKato, Makoto Kawada, Kaori Kuroda and Yuko Okazima for sample collections. \nAuthor contributions: Kyoko Nishikimi and Takako Kiyokawa conceived and carried out \nexperiments; Kyoko Nishikimi and Shinichi Tate analysed data; Takako Kiyokawa and \nMasami Iwamoto confirmed the histology and evaluated the immunohistochemistry; and \nShinichi Tate and Makio Shozu collected clinical data and specimens. All authors were \ninvolved in writing the paper and approve of the submitted and published versions. \n \n\nReferences \n[1] Wang DD, Chen YB, Pan K, et al. Decreased expression of the ARID1A gene is \nassociated with poor prognosis in primary gastric cancer. PLOS ONE 2012; 7(7): e40364. \n[2] Wiegand KC, Shah SP, Al-Agha OM, et al. ARID1A mutations in endometriosis-\nassociated ovarian carcinomas. N Engl J Med 2010; 363: 1532-1543. \n[3] Jones S, Wang TL, Shih IeM, et al. Frequent mutations of chromatin remodeling gene \nARID1A in ovarian clear cell carcinoma. Science 2010; 330: 228-231. \n[4] Wu RC, Wang TL, Shih IeM. The emerging roles of ARID1A in tumor suppression. \nCancer Biol Ther 2014; 15(6): 655-664.  \n[5] Wu JN, Roberts CW. ARID1A mutations in cancer: another epigenetic tumor suppressor? \nCancer Discov 2013; 3(1): 35-43.  \n[6] Ho L, Crabtree GR. Chromatin remodelling during development. Nature 2010; 463: 474-\n484. \n[7] Wang X, Nagl NG, Wilsker D, et al. Two related ARID family proteins are alternative \nsubunits of human SWI/SNF complexes. Biochem J 2004; 383: 319-325.  \n[8] Van Rechem C, Boulay G, Leprince D. HIC1 interacts with a specific subunit of \nSWI/SNF complexes, ARID1A/BAF250A. Biochem Biophys Res Commun 2009; 385: 586-\n590. \n[9] Guan B, Rahmanto YS, Wu RC et al. Roles of deletion of Arid1a, a tumor suppressor, in \n\nmouse ovarian tumorigenesis. J Natl Cancer Inst 2014; 106: (7) pii: dju146. \n[10] Chandler RL, Damrauer JS, Raab JR et al. Coexistent ARID1A-PIK3CA mutations \npromote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine \nsignalling. Nat Commun 2015; 6: 6118. \n [11] Maeda D, Mao TL, Fukayama M, et al. Clinicopathological significance of loss of \nARID1A immunoreactivity in ovarian clear cell carcinoma. Int J Mol Sci 2010; 11(12): 5120-\n5128. \n[12] Yamamoto S, Tsuda H, Takano M, Hase K, Tamai S, Matsubara O. Clear cell \nadenofibroma can be a clonal precursor for clear cell carcinoma of the ovary: a possible \nalternative ovarian clear cell carcinogenic pathway. J Pathol 2008; 216: 103-110. \n[13] Yamamoto S, Tsuda H, Suzuki K, Takano M, Tamai S, Matsubara O. 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Loss of ARID1A expression is an early molecular \nevent in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid \ncarcinoma. Int J Gynecol Cancer 2012; 22(8): 1310-1315.  \n\n[22] Xiao W, Awadallah A, Xin W. Loss of ARID1A/BAF250a expression in ovarian \nendometriosis and clear cell carcinoma. Int J Clin Exp Pathol 2012; 5(7): 642-650. \n[23] Yamamoto S, Tsuda H, Takano M, Tamai S, Matsubara O. Loss of ARID1A protein \nexpression occurs as an early event in ovarian clear cell carcinoma development and \nfrequently coexists with PIK3CA mutations. Mod Pathol 2012; 25(4): 615-624. \n[24] Katagiri A, Nakayama K, Rahman MT, et al. Loss of ARID1A expression is related to \nshorter progression-free survival and chemoresistance in ovarian clear cell carcinoma. Mod \nPathol 2012; 25(2): 282-288. \n \n  \n\nTable 1. Clinicopathological features of 93 ovarian clear cell carcinomas and loss of \nBAF250a expression (#1NX, lymphadenectomy not performed) \nParameters n Loss of BAF250a expression (%) \nTotal 93 50 (54)  \nMean age [years (range)] 56 (32–84)  \nTumour infiltration   \nT1a 27 11 (41) \nT1b 1 1 (100) \nT1c 58 33 (57) \nT2a 0 0 (0) \nT2b 0 0 (0) \nT2c 5 3 (60) \nT3a 1 1 (100) \nT3b 0 0 (0) \nT3c 1 1 (100) \nLymph node metastasis   \nN0 51 31 (61) \nN1 1 0 (0) \nNX#1 41 19 (46) \nReceived adjuvant chemotherapy   \nYes 80 48 (60) \nNo 13 2 (15) \nRecurrence   \nYes 20 12 (60) \nNo 73 38 (52) \n\nTable 2. Loss of BAF250a expression in carcinoma, adenofibroma, and endometriosis \n   Loss of BAF250a expression \nAssociated lesion No. of cases   Adenofibroma Endometriosis Carcinoma \nAdenofibroma+/Endometriosis+ 18  0 (0%) 0 (0%) 8 (44%) \nAdenofibroma+/Endometriosis- 18  0 (0%) - 5 (28%)* \nAdenofibroma-/Endometriosis+ 45  - 0 (0%) 30 (67%)* \nAdenofibroma-/Endometriosis- 12   - - 7 (58%) \n*p = 0.01, Fisher’s exact test \n  \n\nTable 3. Univariate and multivariate analysis of loss of BAF250a expression in carcinoma \nassociated with adenofibroma and endometriosis \n \n \n \n \n \n \n \n \n \n \n \n \n        Univariate analysis   Multivariate analysis \nAssociated \nlesion  \nNo. of \ncases \nNo. of cases \nwith loss of \nBAF250a \nexpression in \ncarcinoma \n  \nOdds \nratio \n95% CI p-value   \nOdds \nratio \n95% CI p-value \nAdenofibroma+ 36 13 (36%)  0.3 0.1–0.8 0.01  0.3 0.1–0.9 0.03 \nEndometriosis+ 63 38 (60%)   0.6 0.2–1.1 0.10   1.8 0.7–4.6 0.25 \n\nFigure 1. Representative histological features (A–D; hematoxylin and eosin staining) and \nBAF250a immunostaining (E–H) of clear cell carcinoma (CCC) and associated adenofibroma \nor endometriosis (×100). (A,B) CCC associated with adenofibroma. (C,D) CCC associated \nwith endometriosis. (E) BAF250a expression is retained in both CCC and adenofibroma. (F) \nBAF250a expression is lost in CCC but retained in adenofibroma. (G) BAF250a expression is \nretained in both CCC and endometriosis. (H) BAF250a expression is lost in CCC but retained \nin endometriosis. \n  \n\n\nFigure 2. Kaplan–Meier curves of progression-free survival and BAF250a expression in \npatients with pT1 cancer. \n \n  \n\n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nHistopathology Vol.67 Issue 6 p.866-871 \n平成 27 年6 月7 日 公表済","source_license":"CC0","license_restricted":false}