{"paper_id":"041f54ee-1689-454c-9738-0b14f2ff7d8d","body_text":"Submit Manuscript | http://medcraveonline.com\nAbbreviations: OE, ovarian endometriosis; HNF-1β, \nHepatocyte nuclear factor-1β; H-score, Histological score\nIntroduction\nEndometriosis represents one of the most crucial problems in \nmodern gynecology. It is a multifactorial condition characterized \nby the pathological growth of tissue resembling the endometrium \nin its morphological and functional properties, outside the lining \nof the uterus. 1 In 2-22% of cases, endometriosis is diagnosed in \nthe general population following asymptomatic course, in 50-60% \nof cases, in patients presenting with pelvic pain, and in 20-30%, in \npatients with infertility. In postmenopausal women, endometriosis \nis diagnosed in 2-5% of cases. 2–5 The first clinical observations \nsupporting the association between endometriosis and ovarian cancer \ndate back to almost a century ago. 6 However, it was only in the late \n20th century that large-scale epidemiological studies confirmed the \n2.5-fold increase in risk of cancer over a 10-year follow-up period in \nwomen with endometriosis.7 Endometriosis does indeed share certain \ncharacteristics with malignant conditions, such as the presence of \nboth localized and distant foci, and the propensity for cell migration \nand invasion resulting in target organ damage. 8,9 The histogenesis of \novarian endometriosis (OE) and endometriosis-associated tumors \nremains a matter of dispute. A 2.5-fold increase in the risk of malignant \novarian tumors was shown in OE. 10 Foci of endometrial epithelial \nmetaplasia are observed in 12.1% of OE cases, foci of hyperplasia \nin 9.4%, foci of atypia in 5.9%, while ovarian cancer is diagnosed \nin 4.1% of such cases. 11 Other studies reported that the prevalence \nof epithelial atypia foci in OE may reach 35-80%. 12,13 Ovarian cancer \nrepresents a morphologically and biologically heterogenous group of \ntumors. Serous tumors are characterized by the mutation in p53 gene, \nmucinous tumors - by the mutation in K-ras gene, and endomethrioid \ntumors - by those in beta-katenine and PTEN genes.14\nThe protein p53 regulates the cell cycle, apoptosis, DNA reparation \nand suppresses the transcription of the Bcl-2 gene. Mutations in the \np53 gene which may occur at any stage of the malignant transformation \nincrease the expression of p53 protein and the risk of various \nactions of the mutant protein on its “targets” (protein expression is \nincreased in foci of dysplasia as compared to normal tissues). Вcl-\n2 is a mitochondrial oncoprotein, an apoptosis inhibitor, a member \nof the Bcl-2 (B-cell lymphoma 2 protein)  family. The effect of this \nprotein is exerted in the phase of DNA degradation which represents \nthe terminal stage of the process of apoptosis regulation. 15 Ki-67 is \nthe marker of cell proliferation, which is relatively as informative \nas the 3H-thymidine label. The expression of Ki-67, which is a non-\nhistone nuclear protein, allows identify proliferating cells in the late \nG1, S, M and G2 phases of the cell cycle, with the exception of G0 \nand early G1 phases, since this protein is degraded within 60 to 90 \nInt J Fam Commun Med. 2018;2(2):75‒78. 75\n© 2018 Levakov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which \npermits unrestricted use, distribution, and build upon your work non-commercially.\nEnhanced expression of some biomarkers as a \npredictive factor for malignant transformation of \nlesions with atypia in ovarian endometriosis\nVolume 2 Issue 2 - 2018\nSergey A Levakov, Tatiana A Gromova\nDepartment of Obstetrics and Gynecology, First Moscow \nMedical State University, Russia\nCorrespondence: Sergey A Levakov, Department of \nObstetrics and Gynecology, First Moscow Medical State \nUniversity, 8/2 Trubetskaya Str, Moscow 119048, Russian \nFederation, T el +7(925)5063144, Email levakoff@yandex.ru \nReceived: April 12, 2018 | Published: April 24, 2018\nAbstract\nBackground: Patients with ovarian endometriosis (OE) are known to have a 2.5-fold \nincrease in the risk of malignant neoplasms. OE is polymorphic condition histologically \nsubdivided on two forms: “without atypia” and “with atypia”. First form is considered \nbenign, while second one is precursor of ovarian malignant transformation. \nAim: Assess of the morphological and immunohistochemical characteristics indicative of \nthe risk of malignant neoplastic transformation of OE atypical lesions.\nMaterials and Methods: Seventy-eight fragments of ovarian tissue (resected regarding \novarian endometrioid cists) were studied histologically for determination of cystic ovarian \nendometriosis foci with and without atypia. Thirty-five histological slides with OE and \n8 slides with adenocarcinomas of different types were studied immunohistochemically. \nMonoclonal antibodies to Ki-67, Всl-2, р53 proteins and polyclonal antibodies to HNF-1β \nwere used. Results were evaluated by means of Histological score (H-score) method. \nResults: 35 foci of cystic OE without atypia and 32 foci with epithelial atypia were \nobserved. In foci with epithelial atypia, the expression of Ki-67, Bcl-2 and HNF-1β was \nsignificantly higher relatively to the foci without atypia, while expression of р53 was the \nsame in foci with and without atypia. From 4 subtypes of studied ovarian adenocarcinomas \nthe expression of HNF-1β was only observed in clear cell adenocarcinoma.\nConclusion: Enhanced expression of biomarkers Ki-67, Bcl-2 and HNF-1β in cystic OE \nwith atypia indicates on the risk of malignant transformation of lesions with atypia. Using \nof these biomarkers can be useful for differential diagnostics of OE with and without atypia \nand for decision about surgical treatment. \nKeywords: ovarian endometriosis, endometriosis with atypia, ovarian tissue, epithelial, \nInternational Journal of Family & Community Medicine \nResearch Article\n Open Access\n\n\nEnhanced expression of some biomarkers as a predictive factor for malignant transformation of lesions \nwith atypia in ovarian endometriosis\n76\nCopyright:\n©2018 Levakov et al.\nCitation: Levakov SA, Gromova TA. Enhanced expression of some biomarkers as a predictive factor for malignant transformation of lesions with atypia in \novarian endometriosis. Int J Fam Commun Med. 2018;2(2):75‒78. DOI: 10.15406/ijfcm.2018.02.00051\nminutes after mitosis. A correlation between the level of expression \nof Ki-67 and the level of mitotic activity of the cells has been clearly \ndemonstrated.16 Hepatocyte nuclear factor-1β (HNF-1β) is a protein \nof the transcription factors super family which binds to the DNA in \nthe form of a homo- or heterodimer. It has been considered a specific \nimmunohistochemical marker of the clear cell ovarian tumors which \nare most often associated with OE. Other tumors of the ovaries \nand the endometrium do not express HNF-1β. 17–22 HNF-1β plays a \ncrucial role in protecting cells against oxidative stress, suppresses \napoptosis by means of interaction with apoptosis inhibitor Bcl-2. \nThe expression of HNF-1β was observed in the epithelium of 40% \nof ovarian endometrioid cysts and is more often observed in cases of \natypia of the epithelium in endometrioid lesions. \nMaterials and methods\nIn this retrospective study, we included 78 fragments of ovarian \ntissue retrieved following ovarian resection due to endometrioid \ncysts, and 8 ovarian adenocarcinomas (2 clear cell, 2 well-\ndifferentiated serous, 2 endometrioid and 2 mucinous). Patients with \nadenocarcinomas had no history of OE or signs of OE in retrieved \nsamples. For the histological investigation of the 78 cases, a standard \ntechnique was employed using 10% neutral buffered formalin, \nparaffin embedding and histological section of 3 µm  from paraffin \nblocks in НМ355S rotary microtome (Thermo Scientific, Germany). \nThe sections were further spread on glass slides and hematoxylin-\neosin stained. The assessment of sections allowed confirm and \nspecify the pathohistological diagnosis, to identify endometriotic \nlesions (apart from the endometriotic cyst which was the reason  for \nsurgery) in ovarian tissue, and to select paraffin blocks to perform the \nimmunohistochemical assessment. OE was confirmed using standard \nmorphological criteria, i.e. the presence of epithelial lining of the cyst \nand/or of glandular components consisting of endometrioid cells (with \naccount of reactive, regeneratory, dystrophic, metaplastic changes, \nand atypia), the presence of endometrioid stroma and macrophages \nwith hemosiderine contained in the infiltrate and/or cyst lumen. 23–25 \nForty three samples were assessed using the immunohistochemical \nmethod - of them, 35 were OE samples (assessed using four mono- and \npolyclonal antibodies) and 8 samples were ovarian adenocarcinomas: \n2 clear cell, 2 well-differentiated serous, 2 endometrioid and 2 \nmucinous. Patients with adenocarcinomas had no history of OE or \nsigns of OE in retrieved samples. \nThe four types of adenocarcinomas were assessed for determination \nof specificity of HNF-1β expression in ovarian tumors. As primary \nantibodies, we used the antibodies to a cell proliferation marker, \nnuclear protein Ki-67 (monoclonal antibody, MIB-1 clone, DAKO, \nNorway, at a dilution of 1:150), apoptosis inhibitor Bcl-2 (monoclonal \nantibody, clone 124, Cell Marque, USA, at a dilution of 1:250), \noncoprotein p53 (monoclonal antibody, clone DO7, Cell Marque, \nUSA, RTU dilution), and transcription factor super family protein, \nhepatocyte nuclear factor-1beta (HNF-1β, polyclonal antibody, \nGeneTex, USA, at a dilution 1:200). The study was performed \nusing serial 3µm sections produced from paraffin blocks selected \nin course of preliminary histological assessment of all available \ntissue specimens from each of the cases. The sections were placed \non special covered glass slides for staining (endogenous peroxidase \nin deparaffinated slides was blocked using 3% peroxide). Chemical \nreactions were performed simultaneously for the complete set of \nspecimens in order to obtain comparable data. The sections were \ndeparaffinized on glass with of xylene and further washed with 5 \nrinses of ethanol in descending concentrations and then with distilled \nwater, following the protocol described in Table 1. We further applied \nhigh-temperature antigen retrieval following the technique described \nby SR.Shi et al. 26,27 via the incubation of histological sections in a \nbuffer solution (citrate, pH 6.2, or TRIS-EDTA, pH 8.0, subject to \nrecommendations provided by primary antibody manufacturers) in \na RT Module (Thermo Scientific, Great Britain) for heat-induced \nprocessing of formalin fixed paraffin-embedded tissue sections. The \nfollowing settings were selected: temperature 98-99 оС, incubation \ntime 20±1min, pre-heating 65оС. \nT able 1 Histological sections deparaffination protocol\nRinse № Solvents T emperature Duration (min)\n1 Xylite I 20-22°C 10-15min\n2 Xylite II 20-22°C 5-10min\n3 Xylite III 20-22°C 2min\n4 Ethanol \nabsolute 20-22°C 2min\n5 Ethanol 96% I 20-22°C 2min\n6 Ethanol 96% II 20-22°C 2min\n7 Ethanol 70% 20-22°C 2min\n8 Ethanol 40% 20-22°C 2min\n9 Distilled water 20-22°C 5min\nSamples cooled to 85оС were transferred on tripods into the unit for \nimmunohistochemical and immunocitological staining, (Autostainer, \nThermo Scientific, Great Britain). Immunohistochemical reactions \nwere performed in accordance with the protocols provided by \nmanufacturers for mono- and polyclonal antibodies. For reaction \nvisualisation, we used a ready test-system with universal secondary \nchromogen (3,3’ - diaminobenzydine)-labeled antibodies Histophine \n(Nichirei Corp., Japan). Two negative controls (for reaction specificity \nand absence of active endogenous peroxidase) and one internal \npositive control (for reaction specificity) were accounted for. Results \nwere evaluated by assigning the Histochemical score (H-score) \nmethod 28,29 based on the number and intensity of stained cell nuclei \n(Ki-67, p53, HNF-1β) and cytoplasm (Bcl-2): score below 80 was \ninterpreted as low, 80-140 as moderate, and 141-300 as high staining \nintensity. The evaluation was performed for 100 epithelial cells \nin three randomly selected fields of view with x400 magnification. \nThe statistical analysis was performed using Statistica for Microsoft \n10 software. Mean and the standard deviation were reported for the \ndate sample and a non-parametric Wilcoxon-Mann-Whitney test was \nperformed with a critical value of 0.05 (p<0.05). \nResults\nOf the 78 assessed cases, in the epithelium of endometrioid cyst \nwalls and/or other endometriotic lesions, the following microfoci \nor larger foci were observed: with syncytial papillary regenerative \n(hyperplastic) changes - 31 cases (39.7%), with hobnail-cell \nmetaplasia in 12 cases (15.4%), with focal hyperplasia (papillary, \nmixed glandular and solid, with squamous metaplasia or with loci \nof squamous metaplasia) - 3 cases (3.9%). In 32 cases (41.0%), \nepithelial atypia was identified which in 27 cases (34.6%) was \nestimated as “regenerative/dystrophic”,23,24 due to marked dystrophia \nof epitheliocytes combined with the accumulation of intraepithelial \nleucocytes and the inflammatory infiltration of adjacent stroma of \n\nEnhanced expression of some biomarkers as a predictive factor for malignant transformation of lesions \nwith atypia in ovarian endometriosis\n77\nCopyright:\n©2018 Levakov et al.\nCitation: Levakov SA, Gromova TA. Enhanced expression of some biomarkers as a predictive factor for malignant transformation of lesions with atypia in \novarian endometriosis. Int J Fam Commun Med. 2018;2(2):75‒78. DOI: 10.15406/ijfcm.2018.02.00051\nthe endometrioid lesion, while in 5 cases (6.4%) such changes were \nabsent and “true neoplastic” atypia was diagnosed. In 1 case (1.3%), \na small focus of a borderline mucinous tumor was found in the wall \nof an endometrioid cyst. In 43 of the 78 histologically assessed OE \ncases (55.1%), within the resected ovarian fragments containing an \nendometrioid cyst, endometriotic lesions (from 1 to 4) were identified. \nThose were presented with epithelial and stromal components both \nwithin the cyst walls and at varying distance from it. Summarizes of \nthe results of immunohistochemical assessment of the selected OE \nand 8 ovarian adenocarcinoma cases are described in the Table 2. As \nit is seen from the table, in foci with epithelial atypia, the expression \nlevel of Ki-67, Bcl-2 and HNF-1 β was significantly higher relatively \nto the foci without atypia. The differences in these marker proteins \nexpression between endometriosis foci with atypia and without atypia \nare statistically significant (p <0.05). Expression level of р53 protein \nwas low in comparison with levels of three other studied proteins and \nno significant difference in p53 expression was revealed between OE \nfoci with and without atypia (p >0.05).\nT able 2 Results of immunohistochemical determination of expression level of \nbiomarkers Ki-67, p-53, BCL-2 and HNF-1β in foci of ovarian endometriosis \nwith atypia and without atypia \nMarker protein\nOE foci without atypia\nH-score: M±σ\nn=35\nOE foci with atypia\nH-score: M±σ\nn=32\nKi-67 27±14 79±11*\np53 12±10 16±12\nBcl-2 43±11 121±27*\nHNF-1B 62±17 188±13*\nNote: n=35 is the number of OE foci without of atypia; n=32 is the number \nof OE foci with atypia. The OE foci were selected from 35 ovarian fragments. \nEach of the fragments contained the foci both with and without atypia, with \nexception of 3 fragments, which did not contain foci with atypia. *- means \nthat the difference in expression level of the biomarker in foci with atypia \ncompared to foci without atypia is statistically significant (p<0.05)\nIt should be noted, that unlike OE foci with atypia, foci with \nsyncytial papillary regenerative (hyperplastic) changes, with hobnail-\ncell metaplasia, and focal hyperplasia (papillary, mixed glandular and \nsolid, or with loci of squamous metaplasia) did not differ from normal \nendometrial epithelium in the expression of Ki-67, p53 or Bcl-2. The \nexpression of the specific transcription factor of clear cell ovarian and \nendometric tumors HNF-1β was observed in epithelial nuclei of the \nmajority of endometrioid lesions. It appeared in the nuclei of both \nendometrioid cysts epithelium and other endometrioid epithelium \nlesions (those found in walls of cysts and at a distance from them) \nregardless of the presence of metaplastic or hyperplastic changes \nin the epithelium. The evaluation of the number of endometrioid \nlesions with varying morphological features demonstrated that in \nOE foci without atypia in 56 % of cases the expression of HNF-\n1β was observed, while in OE foci with atypia (regenerative/\ndystrophic or true) HNF-1β was expressed in 94% of epithelial \nnuclei. Immunohistochemical assessment of HNF-1β expression \nin clear cell (n=2), well-differentiated serous (n=2), endometrioid \n(n=2) and mucinous (n=2) adenocarcinomas showed that HNF-1β is \nindeed enough specific marker for clear cell adenocarcinoma.23 Other \nassessed ovarian tumors actually do not express HNF-1β (only a few \nsingle cells with mild intensity of nuclei staining). In the borderline \nmucinous tumor found in the wall of an endometrioid cyst in one \ncase the expression of HNF-1β was unidentifiable. Expression level \nof HNF-1β in clear cell appeared significantly higher in comparison \nwith the expression level in OE foci without atypia: 195±29 vs 62±17 \nH-score (the difference is statistically significant, p<0.05) and was, in \nfact, equal to the expression level in OE foci with atypia (195±29 vs \n188±13 H-score, p>0.05). \nConclusion\nThe results of this study confirmed the high prevalence of \nhistological, as well as molecular-biological changes in OE lesions \nsuggesting an increased risk of neoplastic transformation. OE lesions \nwith histological signs of epithelial atypia (regenerative/dystrophic \nin 34.6% and true neoplastic in 6.4%) were characterized by \nincrease expression of the proliferation marker Ki-67, the apoptosis \ninhibitor Bcl-2 and the specific transcription factor of clear cell \novarian and endometric tumors HNF-1β. The observed coincidence \nof expression levels of HNF-1β in OE lesions with atypia and in \nclear cell adenocarcinoma, while the both levels are significantly \nhigher of expression level of HNF-1β in OE lesions without atypia \n(see Table 2), indicates on counteraction of pathogenesis pass ways \nof OE lesions with atypia and clear cell ovarian adenocarcinoma. It \nwas reliable established, that OE most often associated with clear \ncell adenocarcinoma. Quite probably, that at least part of clear cell \nadenocarcinomas arise as a result of malignant transformation of OE \nlesions with atypia.30 Using of immunohistochemical markers Ki-67, \nBcl-2 and HNF-1β, which enhance their expression in OE lesions with \natypia may be a useful approach for differential diagnostics of OE \nsubtypes with and without atypia and preventive surgical treatment of \npatients with OE. \nAcknowledgements\nNone.\nConflict of interest\nThe authors declare there is no conflict of interest.\nReferences\n1. 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