{"paper_id":"b9e210c3-acec-41da-aebb-d69f4492067b","body_text":"J of IMAB. 2019 Jul-Sep;25(3) https://www.journal-imab-bg.org 2611\nABSTRACT\nPurpose: The aim of the study was to establish a\nloss of ARID 1A protein expression in cases of ovarian\nendometriomas and its probable role in the development\nof clear-cell and endometrioid ovarian carcinomas.\nMaterial and Methods: The immunohistochemical\nanalysis of ARID 1A protein expression was performed on\nspecimens collected from the study group (group 1) that\nincluded 72 patients with endometrioid ovarian cysts. The\ncontrol group (group 2) included 15 patients with clear-\ncell and endometrioid ovarian carcinomas.\nResults: In the study group, 1 of 72 specimens\n(1,4%) showed a complete absence of reactivity and was\ndefined as ARID 1A protein deficient. In the control group,\n5 of 15 specimens (33.3%) were found to be ARID 1A pro-\ntein deficient. In 7 (46.7%) cases of this group, together\nwith the malignant component adjacent endometriosis was\ndiagnosed. Two of these endometrioses were atypical, with\nan ARID 1A protein deficient expression.\nConclusions: These data confirm the hypothesis\nthat in some endometriomas mutation of the tumor-sup-\npressor ARID 1A gene occur, leading to the loss of pro-\ntein expression and its functional activity, thus indicat-\ning a high risk for the development of endometriosis-as-\nsociated ovarian cancer.\nKey words:  ARID 1A, endometrioma, endometrio-\nsis-associated ovarian carcinomas (EAOCs), ovarian clear-\ncell carcinoma (OCCC), endometrioid ovarian carcinoma\n(EnOC)\nINTRODUCTION\nEndometriosis is an estrogen-dependent benign\ncondition that affects 5-10% of females in reproductive\nage [1, 2]. It is defined as the presence of endometrial\nglands and stroma outside the endometrial mucosa and\nuterine musculature [2]. In 1925 Sampson first described\na malignant transformation of endometriosis into ovarian\ncancer. Since then, much evidence has been accumulated\nfor the connection between endometriosis with two types\nOriginal article\nLOSS OF ARID 1A PROTEIN EXPRESSION IN\nOVARIAN ENDOMETRIOMAS AS A PROBABLE\nPREDISPOSITION TO DEVELOPMENT OF\nENDOMETRIOSIS-ASSOCIATED OVARIAN\nCARCINOMAS\nTihomir Totev1, Grigor Gorchev 2, Slavcho Tomov 2\n1) Institute of Science and Research, Medical University - Pleven, Bulgaria\n2) Oncogynaecology Clinic, University Hospital, Medical University - Pleven,\nBulgaria.\nJournal of IMAB - Annual Proceeding (Scientific Papers). 2019 Jul-Sep;25(3)Journal of IMAB\nISSN: 1312-773X\nhttps://www.journal-imab-bg.org\nof ovarian cancer: clear-cell and endometrioid [3, 4]. The\ntransition from benign endometriosis to malignant neo-\nplasms is a multifactorial and step-like process. One of\nthe mechanisms of this transformation is related to the\nmutations of the tumor-suppressor gene A T rich interac-\ntive domain 1A (ARID 1A), which is located on the 1ð36\nchromosome. These mutations result in loss of synthesis\nof the protein BRG-associated factor 250a (BAF250a),\nwhich represents a large subunit of the transcription-regu-\nlating human SWI/SNF complexes and play an important\nrole in the control of cell proliferation and tumor suppres-\nsion [5, 6, 7]. Wiegand et al. [5] reported finding muta-\ntions of ARID 1A in 46% of 119 clear-cell ovarian carci-\nnomas, in 30% of 33 endometrioid ovarian carcinomas,\nand none in 76 highly differentiated serous carcinomas.\nIn some endometriomas, genetic and histological changes\noccur, which are an intermediate stage in the malignant\ntransformation. The histological characteristics of these\nintermediate lesions, called “atypical endometriosis” were\ndescribed by Czernobilsky and Morris [8], LaGrenade and\nSilverberg [9]. Areas with atypical endometriosis were\nidentified in 54% of clear-cell and in 42% of endometrioid\ncarcinomas [10]. Additional evidence regarding the im-\nportance of mutations of ARID 1A in the pathogenesis of\nEAOCs are cases, in which loss of protein BAF250a ex-\npression was seen as occurring simultaneously in observed\nARID 1A mutation in a tumour and adjacent atypical en-\ndometriosis [5]. Samartzis et al. [11] found complete ab-\nsence of BAF250a expression in 3 endometriomas (n=3/\n20, 15%), 1 deep infiltrating endometriosis (n=1/22, 5%),\nwhile no such absence was established in peritoneal en-\ndometriosis (n=0/16) and eutopic endometrium (n=0/30).\nThis was why we focused on the ovarian location of en-\ndometriosis as potential precancerosis. Since the immu-\nnohistochemistry of ARID 1A protein expression in\nEAOCs has demonstrated a high-degree correlation with\ngene mutations [5, 12], we used immunohistochemical\nanalysis to detect probable molecular alterations in the\nlesions examined. The aim of our study was to investi-\ngate immunohistochemically the loss of ARID 1A protein\nhttps://doi.org/10.5272/jimab.2019253.2611\n\n2612 https://www.journal-imab-bg.org J of IMAB. 2019 Jul-Sep;25(3)\nexpression in endometrioid ovarian cysts and its probable\nrole in the development of endometriosis-associated ovar-\nian carcinomas.\nMATERIALS AND METHODS\nPatients and specimens\nThe immunohistochemical analysis of ARID 1A\nprotein expression was performed on specimens collected\nfrom the study group (group 1) that included benign en-\ndometriomas, and from the control group (group 2) con-\nsisting of clear-cell and endometrioid ovarian carcinomas.\nThe study group included specimens from 72 patients\nwith endometrioid ovarian cysts, operated on\nlaparoscopically at the St Marina University Hospital –\nPleven during the period 2011-2014. The clinical endome-\ntriosis severity score was defined according to the revised\nAmerican Society for Reproductive Medicine (rASRM)\nscore (rASRM).\nThe control group included 15 patients, of whom\n11 were operated on at St Marina University Hospital –\nPleven, and 4 were operated on at the Clinic of\nOncogynaecology of the University Hospital in Pleven\nduring the period 2012-2014. Of the cases from group 2,\n8 were histologically diagnosed with clear-cell carcinoma\nand 7 – with endometrioid carcinoma. Ovarian carcino-\nmas were staged using the International Federation of\nGynecology and Obstetrics (FIGO) staging system, and the\nhistological subtype and the degree of differentiation were\ndefined according to the WHO classification.\nImmunohistochemistry for ARID 1A Detection\nThe tissue samples were fixed in a 10% solution of\nformaldehyde and embedded in paraffin. Sections stained\nwith hematoxylin /eosin were used for the routine his-\ntopathological investigation. We used a commercially\navailable polyclonal rabbit anti-ARID1A antibody\n(HPA005456; Sigma-Aldrich;diluted 1:200) for ARID1A\nprotein detection. Sections were deparaffinized and boiled\nin a microwave oven at 98\noC, 800 W for 30 min in Dako\nEn vision FLEX Target retrieval solution, low pH (\n0.01mol/l citrate buffer, pH 6.0citrate buffer, pH 6.0). The\nsections were then allowed to cool at room temperature.\nThe activity of endogenic peroxidase was blocked using\n3% hydrogen peroxide. The slides were then incubated\nat 22-24\no C with the primary antibody for one hour and\nwere then treated with a dextran polymer reagent, com-\nbined with secondary antibody and peroxidase (Dako En\nvision FLEX/HRP) for 30 min at room temperature. Spe-\ncific antigen-antibody reactions were visualized using\n0.2% 3,3’ diaminobenzidine tetrachloride in an organic\nsolvent. Counterstaining was performed using Mayer’s\nhematoxylin. Normal (non-neoplastic) endothelial cells,\nfibroblasts and lymphocytes show nuclear ARID 1A im-\nmunoreactivity and serve as a positive internal control\n[13]. Sections without primary antibody served as nega-\ntive controls. All endometrioid cysts of the group 1 and\nEAOCs of  group 2 were defined as ARID 1A intact (im-\nmunoreactive) or ARID 1A deficient (non-immunoreac-\ntive). The specimens with any level of immunoreactivity\npresent were defined as ARID 1A intact. In EAOCs with\nadjacent endometriosis, immunoreactivity of the benign\nand malignant components was evaluated.\nStatistical analysis\nDescriptive method was made.\nRESULTS\nARID 1A immunoreactivity in ovarian endometrio-\nmas\nThe tissue specimens in the study group were col-\nlected from endometrioid ovarian cysts measuring 1-12\ncm in diameter. The mean diameter was 5.2 cm. In this\ngroup were included only histologically benign ovarian\nendometriomas. There were no cases exhibiting the histo-\nlogical characteristics of “atypical endometriosis”. 62 pa-\ntients (86.1%) had stage III (moderate) endometriosis, and\n10 (13.9%) had stage IV (severe) endometriosis. Of the 72\nspecimens, 71 (98.6%) showed well-expressed diffuse im-\nmunoreactivity and were defined as ARID 1A protein in-\ntact. One sample (1.4%) showed a complete absence of\nimmunoreactivity and was defined as ARID 1A protein\ndeficient (Table 1).\nARID 1A immunoreactivity in endometriosis-asso-\nciated ovarian carcinomas\nThe tissue specimens in the control group were col-\nlected from tumors sized 4 to 30 cm in diameter, mean size\n11.3 cm. In the EAOCs group, 8 patients were histologically\ndiagnosed with clear-cell carcinoma, and 7 – with\nendomerioid carcinoma: stage I – 9 patients (60%), stage\nII – 3 patients (20%), stage III – 2 patients (13.3%), and\nstage IV – 1 patient (6.7%). In 7 (46.7%) cases of this group,\ntogether with the malignant component, adjacent endome-\ntriosis was diagnosed. In the tumors with adjacent endome-\ntriosis, immunoreactivity of the benign and malignant\ncomponents was evaluated (Table 2).\nTable 1. Frequency distribution of results from investigating ARID A1 expression\nARID 1A Study group Control group\nN Relative share (%) Sp N Relative share  (%) Sp\nNegative 1 1.4 1.4 5 33.3 12.2\nPositive 71 98.6 1.4 10 66.7 12.2\nAll 72 100.0 15 100.0\n\nJ of IMAB. 2019 Jul-Sep;25(3) https://www.journal-imab-bg.org 2613\nOf the 15 samples, 10 (66.7%) showed different de-\ngrees of diffuse immunoreactivity and were defined as ARID\n1A protein intact. The total absence of reactivity was found\nin 5 specimens, and they were defined as ARID 1A pro-\ntein-deficient. Of 7 endometrioid carcinomas, 2 were ARID\n1A protein-deficient and were combined with adjacent\nThree out of 8 clear-cell carcinomas were ARID 1A\nprotein-deficient, and 2 of these 3 were associated with ad-\njacent benign (non-atypical) endometriosis, also ARID 1A\nprotein-deficient. The other 5 clear-cell carcinomas showed\nTable 2. Frequency distribution of carcinomas combined with endometriosis (control group)\nCombined with endometriosis N Relative share (%) Sp\nNone 8 53.3 12.9\nBenign ARID 1A + 3 20.0 10.3\nBenign ARID 1A - 2 13.3 8.8\nAtypical ARID 1A - 2 13.3 8.8\nAll 15 100.0\natypical endometriosis, also ARID 1A protein-deficient.\nThe other 5 endometrioid carcinomas were found immu-\nnoreactive: in one case the carcinoma was combined with\nnon-atypical endometriosis, also ARID 1A protein-intact,\nand in one case the carcinoma was combined with\ncystadenofibroma with borderline malignancy (Figure 1).\nFig. 1 (a, b, c, d) a. Endometrioid carcinoma (HE) b. EnOC-adjacent atypical endometriosis (HE) c. Endometrioid\ncarcinoma (ARID A1 deficient) d. EnOC-adjacent atypical endometriosis (ARID A1 deficient)\nimmunoreactivity, 2 being combined with non-atypical en-\ndometriosis. This component was also ARID 1A protein-\nintact (Figure 2).\n\n2614 https://www.journal-imab-bg.org J of IMAB. 2019 Jul-Sep;25(3)\nFig. 2 (a, b, c, d)  a. Clear-cell carcinoma (HE) b. OCCC-adjacent endometriosis (HE) c. Clear-cell carcinoma\n(ARID A1 focally deficient) d. OCCC-adjacent endometriosis (ARID A1 focally deficient)\nDISCUSSION\nOvarian endometriosis is a benign condition, and its\ntransformation into certain subtypes of cancer is rare [6].\nMany pathogenetic factors in an endometrioma are known\nthat are likely to cause the first molecular alterations in\nDNA in a clone of cells which changes may trigger a ma-\nlignant transformation. The most important of these are\nchronic inflammation and increased level of estrogen,\noxidative stress resulting from the accumulation of heme\nand free iron. It is not known, however, which of the clini-\ncal features of the endometrioma (size, the continuance of\nthe lesion, etc.) are associated with increased risk. Two stud-\nies have pointed to a statistically significant three-fold\nendometriosis [14, 15]. Brinton et al. have reported a risk\nfor ovarian cancer that was 4.2 times higher in a cohort of\n20686 females with ovarian endometriosis of long dura-\ntion [16]. Clear-cell ovarian carcinomas account for 12%,\nand endometrioid carcinomas – for 11% of epithelial ma-\nlignant ovarian tumors [17]. Clear-cell ovarian carcinomas\ndemonstrate a behaviour different from that of the most\ncommon serous tumors: they are comparatively more resist-\nant to conventional chemotherapy with taxane and plati-\nnum-based cytostatic drugs and have a poorer prognosis\nin advanced stages, especially in cases of insufficient sur-\ngical cytoreduction [18, 19, 20]. Since the role of\nendometrioid ovarian cysts as precursors of a significant\nnumber of EAOCs has been undoubtedly proven, a ques-\ntion arises in which of these cysts there exists a higher risk\nof malignant transformation. Mutations of the tumor-sup-\npressor ARID 1À gene are among the commonest gene mu-\ntations that trigger carcinogenesis, and these mutations can\nbe detected immunohistochemically through the expression\nof the protein encoded in this gene. Our study confirms the\nfacts that have been reported so far: 1. Although rarely\n(1.4%), this phenomenon occurs in some endometriomas,\nfound to be completely histologically benign. This proves\nto be one of the earliest and highly important events in\ncarcinogenesis. 2. In the series we study here were two cases\nin group 2 featuring the histological structure of adjacent\n“atypical endometriosis”, which has been identified as a\nmissing link between benign endometriomas and EAOCs.\nThey were, similarly to the adjacent malignant component,\nARID 1A protein-deficient. 3. The occurrence of EAOCs in\nendometrioma was clearly demonstrated in  group 2 - 47.6%\nof the cases, as was the complete loss of ARID 1A immu-\nnoreactivity in 33.3% of the cases. No statistically reliable\n\n\nJ of IMAB. 2019 Jul-Sep;25(3) https://www.journal-imab-bg.org 2615\n1. Bulun SE. Endometriosis. N Engl\nJ Med . 2009 Jan 15;360(3):268-79.\n[PubMed] [Crossref]\n2. Giudice LC, Kao LC. Endome-\ntriosis. Lancet.  2004 Nov 13-19;\n364(9447):1789-99. [ PubMed]\n3. Ness RB. 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[\nPubMed]\n[Crossref]\n13. Yamamoto S, Tsuda H, Takano\nM, Tamai S, Matsubara O. Loss of\nARID 1A protein expression occurs as\nan early event in ovarian clear-cell car-\ncinoma development and frequently\ncoexists with PIK3CA mutations. Mod\nPatol . 2012 Apr;25(4):615-24\n[\nPubMed] [Crossref]\n14. Rossing MA, Cushing-Haugen\nKL, Wicklund KG, Doherty JA, Weiss\nNS. Risk of epithelial ovarian cancer\nin relation to benign ovarian condi-\ntions and ovarian surgery. Cancer\nCauses Control.  2008 Dec;19(10):\nREFERENCES:\n1357-64. [PubMed] [Crossref]\n15. Worley MJ, Welch WR,\nBerkowitz RS, Ng SW. Endometriosis-\nassociated ovarian cancer: a review of\npathogenesis. Int J Mol Sci.  2013 Mar\n6;14(3):5367-79. [\nPubMed] [Crossref]\n16. Brinton LA, Gridley G, Persson\nI, Baron J, Bergqvist A. Cancer risk af-\nter a hospital discharge diagnosis of\nendometriosis. Am J Obstet Gynecol.\n1997 Mar;176(3):572-79. [\nPubMed]\n17. 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[\nPubMed]\nanalysis can be made because only one patient of the 72\nin the group 1 was found with ARID A1 protein deficient\nexpression, and 5 out of the 15 cases were ARID 1A pro-\ntein deficient in the group 2.\nCONCLUSION\nThe loss of the protein coded by ARID 1A that was\nproved immunohistochemically could be considered as a\nbiomarker indicating an increased risk in cases of\nendometrioid ovarian cysts. The majority of the patients\nwere young females with non-accomplished or partially ac-\ncomplished reproductive functions, undergone organ-sav-\ning surgery. The question whether the immunohistochemi-\ncal investigation of ARID 1A protein expression can be ef-\nfectively used in practice as a screening method in view of\ndesigning an individual approach in observation or further\ntreatment is yet to be elucidated by more studies in multi-\nple centers.\nAcknowledgements\nThe study was supported by a grant from Medical\nUniversity – Pleven, Bulgaria. It was approved by the Com-\nmission for Scientific Research Ethics of Medical Univer-\nsity – Pleven.\n\n2616 https://www.journal-imab-bg.org J of IMAB. 2019 Jul-Sep;25(3)\nCorresponding Author:\nTihomir P . Totev MD, PhD\nSt. Marina University Hospital, Department of Gynecology\nBulgarska aviatsia str., Pleven 5800, Bulgaria. tel. +359 888848326\nE-mail: t.totev@mail.bg\nPlease cite this ar ticle as: Totev T, Gorchev G, Tomov S. Loss of ARID 1A protein expression in ovarian endometriomas\nas a probable predisposition to development of endometriosis-associated ovarian carcinomas. J of IMAB.  2019 Jul-\nSep;25(3):2611-2616. DOI: https://doi.org/10.5272/jimab.2019253.2611\nReceived: 05/12/2018; Published online: 22/07/2019","source_license":"CC0","license_restricted":false}