{"paper_id":"14047272-5bec-424e-b40d-19c5e8dea79c","body_text":"REV .CHIM.(Bucharest)♦70♦No. 4 ♦2019 http://www.revistadechimie.ro 1323\nCD10, CD34 and Ki67 Immunohistochemical Markers Expression\nin Endometriosis and Adenomyosis\nROXANA-CLEOPATRA PENCIU1*, LILIANA STERIU 1, SILVIA IZVORANU 1, IULIA POSTOLACHE 1, ANDREI-ADRIAN TICA 2,\nDIANA MOCANU1, OANA-SORINA TICA 3, VASILE SARBU 4, MARIANA DEACU 5,6, GABRIELA BALTATESCU6,7, IRINA TICA 8,\nLUCIAN PETCU 9, VLAD-IUSTIN TICA 1\n1Ovidius University, Department of Obstetrics and Gynecology, Campus, 1 Universitatii Alley, 900470, Constanta, Romania\n2University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of  Pharmacology, 2 Petru Rares Str., 200349,\nCraiova, Romania\n3University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of Obstetrics and Gynecology, 2 Petru Rares\nStr., 200349, Craiova, Romania\n4Ovidius University Constanta, Department of Surgery,, Campus, 1 Universitatii Alley, 900470, Constanta, Romania\n5Ovidius University Constanta,  Department of Morphopathology, Campus, 1 Universitatii Alley,  900470, Constanta, Romania\n6Department of Clinical Pathology, St. Apostle Andrew Emergency County Hospital, 145 Tomis Blvd., 900591,Constanta, Romania,\n7Ovidius University of Constanta, Faculty of Medicine, Research Center -CEDMOG, 145 Tomis Blvd., 900591,\n8Ovidius University of Constanta, Department of Internal Medicine, Faculty of Medicine, Campus, 1 Universitatii Alley, 900470,\nConstanta, Romania\n9Ovidius University of Constanta,  Department of Statistics, Faculty of Dental Medicine, Campus, 1 Universitatii Alley, 900470,\nConstanta, Romania\nEndometriosis is a benign disease represented by existence of endometrial tissue outside the uterine cavity.\nConsidered in the past a type of endometriosis, adenomyosis is, presently, described as a possible different\nentity, comparative to endometriosis. That is the reason why we decided to study two groups of patients -\none with endometriosis and one with adenomyosis - in order to determine if they are one and the same\ndisease. We included all successive patients admitted and surgically treated in the Emergency Clinical\nHospital Constanta between 2015-2017, and, after applying the selection criteria, we assessed 61 patients\n(group 1) diagnosed with endometriosis - ovarian, cervical, caesarian section scar - and 39 patients (group\n2) with adenomyosis. We studied all patients in terms of age, parity, lesions’ size, admissions’ symptoms,\nchronic symptoms and immunohistochemical markers CD10, CD34, Ki67. We chose there three markers\nbecause of their possible relation to endometriosis and because we were unable to find data regarding the\ncomparison of CD34 or Ki67 expression in endometriosis and adenomyosis and because we did not find\narticles that reported the expression of these three immunohistochemical markers, combined, for either\nendometriosis or adenomyosis. According to our study, it seems that endometriosis and adenomyosis are\ndifferent clinically with regard of age and dysmenorrhea, but there was no statistical difference between the\nstudied immunohistochemical biomarkers’ expression in samples of patients with endometriosis or\nadenomyosis.\nKeywords: endometriosis, adenomyosis, immunohistochemistry, age, dysmenorrhea\nEndometriosis is a benign disease represented by\ndetection of endometrial tissue outside the uterine cavity\n[1]. It can be found anywhere in the peritoneal cavity: on\nthe ovaries, the fallopian tubes, on the peritoneum, the\nuterosacral ligaments, the Pouch of Douglas, the rectal-\nvaginal septum and also in caesarian-section scars,\nlaparoscopy or laparotomy scars, on the bladder, bowels,\ncolon, appendix, and rectum [1].\nAdenomyosis, considered in the past a type of\nendometriosis, is represented by the presence of\nendometrial tissue in the uterine muscle wall. There are\nthree different types of adenomyosis - focal adenomyosis,\nfocal adenomyoma and diffuse adenomyosis. Some of the\ntheories include tissue trauma or some vaginal injury,\nwhich determines inflammation and leads to increased\nmacrophages and cytokines which migrate into the uterine\nmyometrium. Another interesting theory would be\nextension of deep infiltrating endometriosis from outside\ninto the uterine wall [2].\nThere is, still, a very important question: endometrioma\nand adenomyoma are the same entity or are they different?\nThat is the reason why we decided to study two groups\nof patients - one with endometriosis (all types) and the\nother one with adenomyosis. We studied the two groups\nby comparing the immunohistochemistry (IHC) expression\nof CD10, CD34 and Ki67, together with the patients’ age\nand admission symptoms.\nThe IHC marker CD10  is known to be expressed by\nhematopoietic neoplasms like acute lymphoblastic\nleukemia and follicular lymphomas, normal endometrial\nstromal cells and endometrial stromal sarcoma [3].\nThe IHC marker CD34  represents a transmembrane\nphosphoglycoprotein, which was first identified on\nhematopoietic stem and progenitor cells and expresses\nangiogenesis in tissues [3].\nThe IHC marker Ki67 is a cellular proliferation marker\n[3].\nWe decided to study these three markers considering\ntheir relation to endometriosis and to determine if they have\nthe same expression for endometriosis and adenomyosis.\nAnother important reason for this study was that we did\nnot find articles which have studied all the three biomarkers\ntogether for endometriosis and adenomyosis.\n* email: roxanapenciu@yahoo.com, Phone: 0727427621\n\nhttp://www.revistadechimie.ro REV .CHIM.(Bucharest)♦70♦ No. 4 ♦20191324\nExperimental part\nMaterial and methods\nIn our study we included all successive patients admitted\nand surgically treated in the Emergency Clinical Hospital\nConstanta over a period of three years, between 2015-\n2017. Sixty-one patients (group 1) were diagnosed with\nendometriosis - ovarian, cervical, caesarian section scar -\nand 39 patients (group 2) with adenomyosis. We studied\nall patients in terms of age, parity, lesions’ size, admissions’\nsymptoms, chronic symptoms and immunohistochemical\nmarkers CD10, CD34, Ki67.\nAll diagnostics were confirmed by pathology. They were,\nall, reevaluated by two pathologist and representative\nsamples of each patient were selected for immuno-\nhistochemistry. Immunohistochemical tests were\nperformed on four µm-thick sections of formalin-fixed,\nparaffin-embedded tissue blocks of cases included in the\npresent study. After the epitope retrieval, tissue sections\nwere incubated with the following antibodies from Biocare\nMedical (ready-to-use): CD10 (56C6 clone), CD34 (QBEnd\n10 clone) and Ki67 (SP6 clone). We used 3,3’\ndiaminobenzidine (DAB) as chromogen, with brown\nstaining. Sections were finally counterstained with Mayer’s\nHaematoxylin. A positive membrane immunostain of\nstromal cell for CD10 was classified in weak, moderate\nand strong [4]. CD34 immunostain was considered positive\nif a distinctive brown color was present in the membrane\nof the endothelial cells or stromal cells. A positive nuclear\nreaction for Ki67antibody was considered if a brown\nstaining was noticed in more than 5 cells [5].\nExperimental data were analyzed with statistical\nsoftware IBM SPSS Statistics 23. The procedures used\nwere: descriptive statistics (to characterize discrete and\ncontinuous variables defined in the data base), Graphics,\nNonparametrical statistical tests (Chi-squared test for\nassociation, correlation between two category variables,\nin order to calculate, in specific circumstances, the risk/\nchance ratio OR, and Chi-squared test for the comparison\nof two proportions) [6-9].\nResults and discussions\nThe majority of patients from the group with\nendometriosis were between 30- 40 years (34 patients -\n55.7%), while the respective highest incidence of the\npatients in the adenomyosis group was in the 40-50 years\nage interval (30 patients - 76.9%) (table 1). This was not\nunusual, as adenomyosis and endometriosis are\ngynecological diseases that are usually common for\nTable 1\n PATIENTS’ AGE IN THE TWO STUDY GROUPS\nTable 2\nCD10\nIIMMUNOHISTOCHEMICAL\nBIOMARKER’S\nEXPRESSION IN\nSAMPLES FROM\nPATIENTS WITH\nENDOMETRIOSIS OR\nADENOMYOSIS\n\n\nREV .CHIM.(Bucharest)♦70♦No. 4 ♦2019 http://www.revistadechimie.ro 1325\nwomen of reproductive age. The average age for women\nwith adenomyosis is about 40 years [4].\nPatients with endometriosis were younger than patients\nwith adenomyosis. There was a statistical difference\nconcerning the age in the two groups (p < 0.001, Chi-\nSquare Test). Interestingly, there were 5 patients in\nmenopause in the adenomyosis group and two in the other\none.\nThirty patients from group 1 and only 4 from group 2\nwere infertile. There was a statistical correlation between\nfertility and each of the two groups. Infertility was more\nassociated to endometriosis than to adenomyosis (p =\n0.001 < α = 0.05, Chi-Square Test). Our results are\nconcordant with the available data, as approximately 10%\nof women are diagnosed with endometriosis, but usually\nit appears in infertile women (40%) [10, 11].\nThe majority of patients from group 2 (35 patients -\n89.7%) had uterine leiomyomas, while only one patient of\ngroup 1 was diagnosed with the respective condition.\nDysmenorrhea was mostly associated to group 1, with\n42 patients (68.9%), compared with group 2, in which only\n10 patients (25.6%) reported it. There was a statistical\ncorrelation between the two parameters - group and\ndysmenorrhea (p < 0.001 < α = 0.05, Chi-Square Test).\nThere was a calculated possibility of 6.41 times bigger to\nfind a patient with dysmenorrhea in group 1 than in group\n2.\nIn our study, the biomarker CD10 was positive for all the\npatients, in both groups, thus confirming the presence of\nendometrial stromal cells (p = 0.143 > α = 0.05, Chi-\nSquare Test) (table 2, fig.  1). It was moderately positive in\n52 patients (85.2%) of group 1 and 31 patients (79.5%) of\ngroup 2. There was no statistical correlation between the\ndegree of positivity of CD10 and the size of endometriotic\nor adenomyotic lesions.\nOur data were logically relevant and confirm the\ncorrectness of the inclusion process, as the immuno-\nhistochemical marker CD10 is largely expressed by\nstromal endometrial cells located outside the uterus. It can\ncertify the diagnosis of endometriosis [4]. It can be also\nused for women with minimal disease, to confirm the\ndiagnosis [12].\nIHC biomarker CD34 was moderately positive in the\nstromal cells of 18 patients (29.5%) in group 1 and in 7\npatients (17.9%) in group 2. It was negative in 43 patients\n(70.5%) in group 1 and 31 patients (79.5%) in group 2 (table\n3, fig. 2). There was no statistical difference between the\ntwo groups (p = 0.213 >  α = 0.05, Chi-Square Test).\nFig. 1. CD10 immunohistochemical biomarker’s expression (%) in\nsamples from patients with endometriosis or adenomyosis\nTable 3\nCD34 IMMUNOHISTOCHEMICAL BIOMARKER’S EXPRESSION IN PATIENTS WITH ENDOMETRIOSIS OR ADENOMYOSIS\nFig. 2. CD34 immunohistochemical biomarker’s expression in\nsamples from patients with endometriosis or adenomyosis\nImmunohistochemical marker CD34 is used to identify\nendothelial cells. Women with endometriosis have an\nincreased cell proliferation in their endometrium. This\nsuggests that the respective endometrium can implant\nitself and survive in ectopic locations, outside the uterine\ncavity [13].  Meenakshi M et al. observed a phenomenon\nof vascular involvement in adenomyosis. When it is\nwidespread, a neoplastic process may be considered. This\nvascular pattern may sustain a new theory of developing\nadenomyosis from the cells that are intimately situated to\nmyometrial blood vessels, probably multipotential\nperivascular cells [14]. The important relation of CD34 with\n\nhttp://www.revistadechimie.ro REV .CHIM.(Bucharest)♦70♦ No. 4 ♦20191326\nendometriosis and adenomyosis was the reason why we\ndecided to determine if there is a correlation between the\ntwo groups. We could not find in the literature a study\ncomparing the expression of this immunohistochemical\nmarker in-between endometriosis and adenomyosis.\nFor group 1, IHC biomarker Ki67 (in glandular cells) was\npositive in 25 patients (40.9%) and negative in 36 patients\n(59.0%). Patients in group 2 expressed almost the same\nproportion - 13 patients positive (33.4%) and 26 patients\nnegative (66.7%) (table 4, fig. 3). There was no significative\ndifference, on this regard, between the two groups (p =\n0.213 > α = 0.05, Chi-Square Tests) (fig. 3).\nTable 4\n KI67 IMMUNOHISTOCHEMICAL\nBIOMARKER’S EXPRESSION IN\nGLANDULAR CELLS FOR\nENDOMETRIOSIS AND\nADENOMYOSIS\nFig. 3. Ki67 glandular (Ki67 Cg) immunohistochemical biomarker’s\nexpression in samples from patients with endometriosis or\nadenomyosis\nThe biomarker Ki67 in stromal cells was moderate\npositive in 49 patients (80.3%) and negative in 7 patients\n(11.5%) in group 1. In group 2 patients, it was moderate\npositive for 37 patients (94.9%) and negative in one patient\n(2.6%) (table 5, fig. 4). There was no statistical difference\nbetween the two groups for IHC Ki67 in stromal cells (p =\n0.052 > α = 0.05, Chi-Square Tests) (fig. 4).\nJehn-Hsiahn Yang et al. observed that a high Ki67 index\nin immunohistochemistry can be predictive for developing\nadenomyosis [15]. On the other hand, Matsumoto Y et al.\nstudied the bcl-2 gene expression for apoptosis and Ki67\nexpression as a proliferative marker. They observed that\nFig. 4.Ki67 stromal (Ki67 Cs) immunohistochemical biomarker’s\nexpression in samples from patients with endometriosis or\n                                       adenomyosis\nTable 5\nKI67 IMMUNOHISTOCHEMICAL\nBIOMARKER’S EXPRESSION IN\nSTROMAL CELLS SAMPLES\nFROM PATIENTS WITH FOR\nENDOMETRIOSIS AND\nADENOMYOSIS\n\nREV .CHIM.(Bucharest)♦70♦No. 4 ♦2019 http://www.revistadechimie.ro 1327\nKi67 was expressed by glandular epithelium of ectopic\nendometrium, without correlation to menstrual phases. In\nthe secretory phase, this immunohistochemical marker\nwas less expressed by eutopic endometrium in functional\nand basal layers. This is the reason why they concluded\nthat adenomyotic lesions have no origin in the basal\nendometrium [16]. Because of these data we decided to\nstudy the IHC biomarker in both glandular and stromal cells.\nAs for CD34, we could not find in the literature a study\ncomparing the expression of Ki67 immunohistochemical\nmarker in-between endometriosis and adenomyosis.\nNeither could we find data concerning the concomitant\nexpression of CD10, CD34 and Ki67 either for endometriosis\nnor for adenomyosis. This leads to the logical assertion of\nthe non-available comparison of endometriosis and\nadenomyosis regarding the bundle CD10, CD34 and Ki67.\nConclusions\nAlthough, in our study, there were significant clinical\nstatistical differences in-between groups with regard to\nage and dysmenorrhea, immunohistochemical markers\nCD10, CD34 and Ki67 (glandular or stromal) were similarly\nexpressed in the samples from patients with\nendometriosis or adenomyosis.\nWe did not find articles that studied the expression of\nthese three immunohistochemical markers, combined, for\neither endometriosis or adenomyosis. We were, equally,\nunable to find data regarding the comparison of CD34 or\nKi67 expression in endometriosis or adenomyosis.\nAcknowledgments: This research was performed in the Center for\nResearch and Development of the Morphological and Genetic Studies\nof Malignant Pathology from the Ovidius University of Constanþa,\nPOSCCE 2.2.1. Project ID: 1844, code SMIS: 48750, CEDMOG, contract\n627/11.03.2014.\nReferences\n1. BROSENS, I., BENAGIANO, G. History of endometriosis. In: GIUDICE,\nL. C., EVERS JLH, HEALY DL.  Endometriosis Science and Practice: Ed\nBlackwell Publishing Ltd, 2012; p 3-18.\n2.LEYENDECKER, G., WILDT, L. Uterine Peristalsis and the\nDevelopment of Endometriosis and Adenomyosis. In: GIUDICE, L.\nC., EVERS JLH, HEALY DL.  Endometriosis Science and Practice, Ed\nBlackwell Publishing Ltd, 2012, p 200-211.\n3.STUART,L.N.CD34.PathologyOutlines.comwebsite.http://\nwww.pathologyoutlines.com/topic/cdmarkerscd34.html.\n4.SUMATHI, V .P , McCLUGGAGE, W .G. CD10 is useful in demonstrating\nendometrial stroma at ectopic sites and in confirming a diagnosis of\nendometriosis. J Clin Pathol. 2002 May; 55(5): 391–392.\n5.KAHYAOGLU, I., KAHYAOGLU, S., MORALOGLU, O., ZERGEROGLU,\nS., SUT, N., BATIOGLU, S. 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POTLOG-NAHARI, C., FELDMAN, A .L., STRAT TON, P ., KOZIOL,\nD.E., SEGARS, J., MERINO, M.J., NIEMAN, L.K.  CD10 immuno-\nhistochemical staining enhances the histological detection of\nendometriosis. Fertil Steril. 2004 Jul; 82(1):86-92.\n13.WINGFIELD, M., MA CPHERSON, A., HEAL Y , D.L., ROGERS, P .A. Cell\nproliferation is increased in the endometrium of women with\nendometriosis. F ertil Steril. 1995 Aug; 64(2):340-346.\n14.MEENAKSHI, M., McCLUGGA GE, W .G. V ascular involvement in\nadenomyosis: report of a large series of a common phenomenon\nwith observations on the pathogenesis of adenomyosis . Int J Gynecol\nPathol. 2010 Mar; 29(2):117-21.\n15.YANG, J.H, WU, M.Y., CHEN, C.D., CHEN, M.J., Y ANG, Y .S., HO, H.N.\nAltered apoptosis and proliferation in endometrial stromal cells of\nwomen with adenomyosis. Human Reproduction, Volume 22, Issue\n4, 1 April 2007: 945–952.\n16.MATSUMOTO, Y ., IWASAKA, T., YAMASAKI, F., SUGIMORI, H. Apoptosis\nand Ki-67 expression in adenomyotic lesions and in the corresponding\neutopic endometrium. Obstet Gynecol. 1999; 94(1):71. \nManuscript received: 28.11.2018","source_license":"CC0","license_restricted":false}