{"paper_id":"bbf64392-b741-45cb-8fd6-af086df93fca","body_text":"Steroid hormone synthesis by the ovarian\nstroma surrounding epithelial ovarian tumors:\na potential mechanism in ovarian\ntumorigenesis\nLuis Z Blanco Jr 1,5, Elisabetta Kuhn 1, Jane C Morrison 1, Asli Bahadirli-Talbott 1,\nAnne Smith-Sehdev 2 and Robert J Kurman 1,3,4\n1Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA; 2Department of\nPathology, Legacy Health Systems, Portland, OR, USA; 3Department of Gynecology and Obstetrics, The Johns\nHopkins Medical Institutions, Baltimore, MD, USA and 4Department of Oncology, The Johns Hopkins Medical\nInstitutions, Baltimore, MD, USA\nEpithelial ovarian tumors are responsive to steroid hormone stimulation and the ovarian stroma may have a\ndirect role in this process. We evaluated immunohistochemical markers of sex-steroid differentiation and\nsteroidogenesis (calretinin, inhibin, steroidogenic factor 1), steroid enzymes involved in hormone biosynthesis\n(CYP17, CYP19, HSD17 β1, AKR1C3 ), and hormone receptors (estrogen receptor, progesterone receptor, and\nandrogen receptor) in 101 epithelial ovarian tumors and in normal structures implicated in ovarian\ncarcinogenesis (ovarian surface epithelium and cortical inclusion cysts) in an attempt to elucidate this process.\nWe hypothesized that ovarian stroma immediately adjacent to tumors express markers of sex-steroid\ndifferentiation and steroidogenesis and steroid enzymes whereas the epithelium contains corresponding\nhormone receptors. As the findings in seromucinous, endometrioid, and clear cell neoplasms, tumors closely\nassociated with endometriosis, were very similar, these were combined into a group designated ‘endometriosis-\nrelated tumors.’ Significantly increased expression of markers of sex-steroid differentiation and steroidogenesis\nwas found in stroma immediately adjacent to endometriosis-related tumors ( P = 0.003) and mucinous tumors\n(primary and metastatic mucinous tumors were combined because of similar findings) ( P o0.0001) compared\nwith more remote ovarian stroma. In addition, sex-steroid enzymes were increased in stroma adjacent to\nendometriosis-related tumors ( P = 0.02) and mucinous tumors ( P = 0.02) compared with more distant stroma.\nSteroid hormone receptors showed greater expression in epithelium compared with stroma in the endometriosis-\nrelated tumors (P = 0.0009), low-grade serous tumors ( P o0.0001), and high-grade serous carcinoma ( P = 0.0036).\nIn contrast, there was greater expression in stroma compared with epithelium ( Po 0.0001) in mucinous tumors,\nwhich may be due to the fact that they are not derived from müllerian epithelium. In conclusion, our findings\nstrongly support the view that the stroma surrounding epithelial tumors in the ovary is activated to elaborate\nsteroid hormones which may stimulate further neoplastic growth. The precise mechanisms by which this\nprocess might occur are complex and require further investigation.\nModern Pathology (2017) 30, 563–576; doi:10.1038/modpathol.2016.219; published online 6 January 2017\nThe traditional view regarding the origin and\npathogenesis of epithelial ovarian carcinomas is that\nthey develop from the ovarian surface epithelium or\nfrom cortical inclusion cysts through a process of\ninvagination of ovarian surface epithelium. As\ncarcinomas generally resemble the epithelium in\nthe organs from which they are derived, the\nmüllerian phenotype of epithelial ovarian cancers\nhas been difficult to reconcile if their proposed\norigin is from the ovarian surface epithelium, which\nCorrespondence: Dr LZ Blanco Jr, MD, Department of Pathology,\nNorthwestern University Feinberg School of Medicine, Feinberg\nPavilion 7-330, 251 East Huron Street, Chicago, IL 60611, USA.\nE-mail: luis.blanco@northwestern.edu\n5Current address: Department of Pathology, Northwestern\nUniversity Feinberg School of Medicine, Chicago, IL, USA.\nReceived 7 July 2016; revised 7 November 2016; accepted 8\nNovember 2016; published online 6 January 2017\nModern Pathology (2017) 30, 563 –576\n© 2017 USCAP, Inc All rights reserved 0893-3952/17 $32.00 563\nwww.modernpathology.org\n\nis essentially mesothelium. Accordingly, their mül-\nlerian phenotype has been explained by invoking\nmetaplasia of the ovarian surface epithelium to\nmüllerian-type epithelium which then undergoes\nmalignant transformation. Recently, a less complex\nexplanation for the müllerian phenotype of these\ntumors has been advanced by proposing that the\nprecursor lesion of invasive high-grade serous\ncarcinoma is an intraepithelial carcinoma in the\nfallopian tube, so-called serous tubal intraepithelial\ncarcinoma that implants on the ovary.\n1–9 It has also\nbeen suggested that some serous tumors might\ndevelop from cortical inclusion cysts lined by\ntubal-type epithelium derived from the fallopian\ntube at the time of ovulation.\n1 Endometrioid and\nclear cell carcinomas are thought to develop from\nendometriosis which is generally thought to be\nderived from endometrial tissue by retrograde\nmenstruation.\n1–3 An obvious question is why do\nthese tumors have a propensity to develop in the\novary rather than in the organ that is the site of the\nputative precursor lesion.\nThere is evidence that sex-steroid hormones have\na role in ovarian carcinogenesis as demonstrated by\nstudies showing that women on hormone replace-\nment therapy have a significantly increased risk for\novarian cancer.\n10 In addition, cell culture studies\nhave shown that hormones, including estradiol,\nprogesterone, and androgens, may modulate the\ngrowth of epithelial ovarian cancer cells by stimulat-\ning cell proliferation and telomerase expression,\ninducing angiogenesis, and regulating metastasis\nthrough the PI3K/AKT pathway.\n11–16\nIn 1964, Scully described the presence of lutei-\nnized stromal cells immediately adjacent to primary\nand metastatic ovarian tumors and reported\noxidative-enzyme activity in these cells, referring to\nthem as enzymatically active stromal cells. He\npostulated that enzymatically active stromal cells\nstimulated growth of implanted epithelium by local\nsynthesis of steroid hormones.\n17 With the availabil-\nity of reagents that can identify enzymes involved in\nsteroidogenesis, we undertook a morphologic and\nimmunohistochemical analysis of the ovarian stroma\nfrom a variety of epithelial tumors, endometriotic\ncysts, and normal components of the ovary that have\nbeen proposed as possible precursors of ovarian\ncancer (ovarian surface epithelium and cortical\ninclusion cysts) to validate this hypothesis.\nMaterials and methods\nCase Selection\nA total of 101 ovarian tissues (neoplasms and normal\ntissue) and 11 normal fallopian tubes were retrieved\nfrom the pathology files of the Johns Hopkins\nHospital (Baltimore, MD, USA) and Legacy Health\nSystems (Portland, OR, USA) from 1993 through\n2014. All available slides and reports were reviewed.\nTable 1 Primary antibodies, sources and conditions used in this study\nAntibody Symbol Clone Vendor Address Cat no. Dilution Stain type Instrument\nAldo-keto reductase family 1, member C3 AKR1C3 NP6.G6.A6 Sigma-Aldrich St Louis, MO, USA A6229 1:10 000 Manual NA\nAndrogen receptor AR ER179(2) Abcam Cambridge, MA, USA Ab108341 1:400 Manual NA\nCalretinin Polyclonal Cell Marque Hot Springs, AK, USA 232A-78 Predilute Automated BenchMarkXT\nCytochrome P450, family 17 CYP17 3F11 OriGene Rockville, MD, USA TA503442 1:800 Manual NA\nCytochrome P450, family 19 (aromatase) CYP19 H4 AbD Serotec Raleigh, NC, USA MCA2077S 1:800 Manual NA\nEstrogen receptor ER 6F11 Leica Microsystems Bannockburn, IL, USA RTU-ER-6F11 Predilute Automated BenchMarkXT\n17beta-hydroxysteroid dehydrogenase type 1 HSD17β1 Polyclonal Sigma-Aldrich St Louis, MO, USA HPA021032 1:300 Manual NA\nInhibin alpha Polyclonal AbD Serotec Raleigh, NC, USA 0100-0549 1:25 Automated Bond Max\nProgesterone receptor PR 16 Leica Microsystems Bannockburn, IL, USA RTU-PGR-312 Predilute Automated BenchMarkXT\nSteroidogenic factor 1 SF-1 N1665 Invitrogen Carlsbad, CA, USA 434200 1:100 Automated Bond Max\nSteroid hormone synthesis by ovarian stroma\n564 LZ Blanco Jr et al\nModern Pathology (2017) 30, 563 –576\n\nThis study was approved by the Institutional Review\nBoard of the Johns Hopkins Medical Institutions.\nA total of 133 sections were evaluated from the 101\ncases (one to three sections per case). These included\n62 sections from malignant epithelial neoplasms\n(30 high-grade serous carcinomas, 6 low-grade\nserous carcinomas, 11 endometrioid carcinomas,\n5 clear cell carcinomas, 1 mucinous carcinoma,\n3 seromucinous carcinomas, and 6 metastatic carci-\nnomas (3 colorectal and 3 low-grade appendiceal\nmucinous neoplasms), 33 atypical proliferative (bor-\nderline) tumors (19 serous, 6 seromucinous, and 8\nmucinous, including 1 with intraepithelial carci-\nnoma), 5 benign tumors (3 mucinous cystadenomas\nand 2 clear cell adenofibromas), 11 ovarian endome-\ntriosis/endometriotic cysts, 6 cortical inclusion cysts\nlined by tubal-type epithelium, 11 ovarian surface\nepithelium samples, and 5 normal ovaries. In\naddition, 10 sections of normal fallopian tube were\nevaluated.\nImmunohistochemistry\nThe primary antibodies and conditions are shown in\nTable 1. Formalin-fixed, paraffin-embedded sections\nwere immunostained using protocols described\npreviously.18 The reagents used are all commercially\navailable and listed in Table 1 and the tests were\nperformed in accordance with the manufacturer ’s\nrecommendations.\nDescription of the Selected Antibodies\nMarkers of sex-cord differentiation and steroidogen-\nesis included calretinin, inhibin alpha, and steroido-\ngenic factor 1 (SF-1). Markers of sex-steroid hormone\nreceptors included estrogen receptor (ER), progester-\none receptor (PR), and androgen receptor (AR).\nMarkers of enzymes involved in sex steroidogenesis\nincluded CYP19 (cytochrome P450 family 19 or\naromatase), CYP17 (cytochrome P450 family 17 or\n17alpha-hydroxylase), HSD17β1 (17beta-hydroxys-\nteroid dehydrogenase type 1), and AKR1C3\n(aldo-keto reductase family 1 member C3 or 17beta-\nhydroxysteroid dehydrogenase type 5). Table 2\nsummarizes the selected immunohistochemical\ntargets and their functions, while Figure 1 illustrates\nthe specific functions of the sex-steroid enzymes in\nthe steroidogenesis pathway.\nTable 2 Functions of targets of immunohistochemical stains (also see Figure 1)\nTarget Symbol Function\nMarkers of sex-cord differentiation/steroidogenesis\nCalretinin Calcium-binding protein structurally related to inhibin\nInhibin alpha Peptide hormone that is a member of the transforming growth factor-beta\nsuperfamily and has growth promoting activities; produced by ovarian granulosa\ncells to inhibit follicle-stimulating hormone\nSteroidogenic factor 1 SF-1 Transcription factor that binds and activates the promoters of various\nsteroidogenic genes, including aromatase\nMarkers of sex-steroid hormone receptors\nAndrogen receptor AR Receptor for androgen\nEstrogen receptor ER Receptor for estrogen\nProgesterone receptor PR Receptor for progesterone\nMarkers of sex-steroid enzymes\nAldo-keto reductase family 1, member\nC3\nAKR1C3 Also referred to as 17beta-hydroxysteroid dehydrogenase type 5; enzyme that\ncatalyzes the conversion between androstenedione and testosterone\nCytochrome P450, family 17 (17alpha\nhydroxylase)\nCYP17 Enzyme involved in the production of progestins, estrogens and androgens,\nincluding catalyzing the cleavage of the C17 –C20 bond of C21 steroids that is\nessential for the biosynthesis of androgens\nCytochrome P450, family 19\n(aromatase)\nCYP19 Enzyme that catalyzes the last steps of estrogen biosynthesis, converting\ntestosterone to estradiol\n17beta-hydroxysteroid dehydrogenase\ntype 1\nHSD17β1 Enzyme that converts low activity estrone to the more potent estradiol\nFigure 1 Steroidogenesis. Schematic representation of the ster-\noidogenesis pathway of steroid hormone production and some of\nthe major enzymes involved.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\nLZ Blanco Jr et al 565\n\nImmunohistochemical Scoring\nNuclear expression of SF-1, AR, ER, and PR,\ncytoplasmic expression of inhibin, and nuclear\nand/or cytoplasmic expression of calretinin,\nCYP19, CYP17, HSD17β1, and AKR1C3 were con-\nsidered positive when 45% of cells reacted. A\nsemiquantitative scoring system was utilized, taking\ninto account both the proportion of positive cells (5 –\n25% +, 425–50% ++, 450–75% +++, 475% ++++)\nand the intensity of immunostaining [mild +(faint\nimmunoreactivity at low power), moderate ++\n(modest immunoreactivity at low power), or strong\n+++ (immunoreactivity easily appreciable at low\npower)].19 The average proportion of positive cells\nand the average intensity were calculated for each\nmarker. Immunoreactivity and intensity of staining\nwas evaluated in the epithelium, stroma immedi-\nately adjacent to the epithelium (defined as ovarian\nstroma closely applied to the epithelium of the\ntumor, endometriosis or cortical inclusion cysts),\nand stroma that was distant from the epithelium\n(defined as ovarian stroma that was ≥ 10 mm away\nfrom the epithelium). Adjacent endometriosis was\nnot present on sections evaluated for any of the\ntumors such as clear cell and endometrioid carcino-\nmas and therefore only ovarian-type (and not\nendometrial-type) stroma was used for the analysis.\nTo correlate the proportion of positive cells and\nintensity of immunostaining, the values were con-\nverted into composite immunohistochemical scores\nby multiplying the individual scores of proportion of\npositive cells by intensity. For example, a case with +\n++ proportion of positive cells (3 points) and strong +\n++ intensity (3 points) of immunostaining would\nhave an immunohistochemical composite score of 9\n(3 × 3). The average composite immunohistochem-\nical score for each marker were then calculated and\nthe differences of the means (epithelium vs adjacent\nstroma and adjacent vs distant stroma within each\ntype of ovarian lesion, ie epithelium vs adjacent\nstroma in high-grade serous carcinoma) were statis-\ntically analyzed using the Student t-test (two-tailed).\nIn addition, the correlation between specific enzyme\nexpression in the adjacent stroma and the corre-\nsponding hormone receptors in the epithelium and\nstroma within each type of ovarian lesion (ie, CYP17\nand AKR1C3 with AR; CYP19 and HSD17β1 with ER)\nwere evaluated using linear regression. All P-\nvalues o 0.05 were considered statistically\nsignificant.\nResults\nMorphology\nOvarian stromal cells not surrounding follicles and\nstromal cells at a distance from the lesional epithe-\nlium were spindled cells with scant cytoplasm. In\ncontrast, the stromal cells immediately adjacent to\nthe lesional epithelium had variable morphology.\nThose associated with endometriosis/endometriotic\ncysts and benign, atypical proliferative (borderline)\nand malignant neoplasms were composed predomi-\nnantly of lutein or theca-like cells (Figure 2). Lutein-\nlike cells are polygonal cells with abundant eosino-\nphilic or vacuolated cytoplasm and round nuclei,\nwhile theca-like cells are oval or plump spindled-\nshaped cells that are densely cellular near the\nepithelium.20 In some instances there was a mixture\nof the two types of cells. The differences in the\nexpression of markers in adjacent and distant\nstromal cells could be a result of the stromal cell\ntype composition or a transformation of the spindled\ncells into hormone producing lutein or theca-like\ncells. However in our study, as well as in a previous\nreport20 there was no difference in the localization of\nthe antigens despite the varying stromal morphology,\ntherefore we did not distinguish them in our\nFigure 2 Representative hematoxylin and eosin-stained (H&E) sections of ( a) a high-grade serous carcinoma with lutein-like stromal cells\nadjacent to the malignant epithelium and ( b) a mucinous carcinoma with theca-like stromal cells adjacent to the malignant epithelium.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\n566 LZ Blanco Jr et al\n\nTable 3 Immunohistochemical results of markers of sex-cord differentiation and steroidogenesis in ovarian stroma\nAdjacent stroma a composite score b Distant stroma c composite score b P-value of\nadjacent vs\ndistantd\nCalretinin\nInhibin\nalpha\nSteroidogenic factor\n1 Total e Calretinin\nInhibin\nalpha\nSteroidogenic factor\n1 Total\nEndometriosis-related tumors (atypical proliferative\nseromucinous tumor, endometrioid carcinoma, clear\ncell carcinoma, seromucinous carcinoma) n =2 4\n6.46 6.27 10.04 7.63 3.73 4.55 7.55 5.27 0.0038\nLow-grade serous tumors (atypical proliferative serous\ntumor and low-grade serous carcinoma) n =2 5\n3.24 2.15 6.38 3.95 2.71 1.86 3.86 2.81 0.1160\nHigh-grade serous carcinoma n = 30 4.29 4.18 3.61 4.02 2.3 5.7 6.4 4.8 0.2403\nMucinous tumors (atypical proliferative mucinous\ntumor, primary and metastatic mucinous carcinoma)\nn =1 5\n8.5 9.86 10.21 9.52 4.36 4.55 6.45 5.12 o0.0001\naStromal cells immediately adjacent to epithelium. bAverage composite score (proportion × intensity). cStromal cells not immediately adjacent to epithelium (not present in all sections examined).\ndComparison of average total composite score in adjacent stroma and average total composite score in distant stroma. eAverage composite score of all of the markers combined.\nTable 4 Immunohistochemical results of markers of sex-steroid hormone receptors in epithelium and adjacent ovarian stroma\nTumor type\nEpithelium composite score a Adjacent stroma b composite score a P-value of\nepithelium vs\nadjacentc\nEstrogen\nreceptor\nProgesterone\nreceptor\nAndrogen\nreceptor Total d\nEstrogen\nreceptor\nProgesterone\nreceptor\nAndrogen\nreceptor Total\nEndometriosis-related tumors (atypical\nproliferative seromucinous tumor,\nendometrioid carcinoma, clear cell\ncarcinoma, seromucinous carcinoma) n =2 4\n8.55 7.64 3.1 7.17 3.36 4.18 6.7 4.31 0.0009\nLow-grade serous tumors (atypical\nproliferative serous tumor and low-grade\nserous carcinoma) n =2 5\n5.65 7.81 4.85 6.17 0.71 1.63 3.92 1.93 o 0.0001\nHigh-grade serous carcinoma n = 30 7.09 3.22 7.28 5.97 1.78 3.78 5.21 3.72 0.0036\nMucinous tumors (atypical proliferative\nmucinous tumor, primary and metastatic\nmucinous carcinoma) n =1 5\n0.14 0.86 0.5 0.5 3.57 5.07 6.33 4.93 o 0.0001\naAverage composite score (proportion × intensity). bStromal cells immediately adjacent to epithelium. cComparison of average total composite score in epithelium and average total composite score in\nadjacent stroma using t-test.dAverage composite score of all of the markers combined.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\nLZ Blanco Jr et al 567\n\nanalysis. Metastatic tumors and some malignant\novarian neoplasms had desmoplastic stroma com-\nposed of fibroblastic-like spindled cells with pale\ncytoplasm. Desmoplastic stroma was distinguished\nfrom luteinized stroma and was negative for all\nmarkers tested.\nImmunohistochemical Findings\nPreliminary analysis revealed some correlation\nbetween the markers in the ovarian stroma and in\nthe epithelium depending on the specific ovarian\nlesions and tumors. Given these findings and based\non known and reported clinicopathologic, immuno-\nhistochemical, and molecular data on the general\ngroups of ovarian epithelial tumors and ovarian\ncarcinogenesis which has been recently\nsummarized,\n21 we grouped the tumors and per-\nformed statistical analysis in four categories as\nfollows: (1) endometriosis-related tumors (atypical\nproliferative (borderline) seromucinous tumors,\n22–26\nclear cell carcinoma, endometrioid carcinoma and\nseromucinous carcinoma), (2) low-grade serous\ntumors (atypical proliferative (borderline) serous\ntumors and low-grade serous carcinoma) (3) high-\ngrade serous carcinoma, and (4) intestinal-type\nmucinous tumors (atypical proliferative (borderline)\nmucinous tumors, primary, and metastatic\nmucinous carcinoma). Primary and metastatic muci-\nnous carcinomas, classified accordingly based on the\ncriteria in the 2014 WHO Classification of Tumours\nof the Female Reproductive Organs,\n27 exhibited\nsimilar immunohistochemical profiles and\nbecause of the small number of cases were analyzed\ntogether in one group. A summary of the results\nindicating the immunohistochemical composite\nscores for the various markers are presented in\nTables 3, 4 and 5. Representative images are shown\nin Figures 3, 4 and 5.\nMarkers of Sex-Steroid Differentiation and\nSteroidogenesis (Calretinin, Inhibin, and Steroidogenic\nFactor 1)\nThere was a statistically significant difference in the\ncomposite score for calretinin, inhibin and SF-1\ntogether in the adjacent vs the distant stroma in\nendometriosis-related tumors ( P = 0.0038) and\nTable 5 Immunohistochemical results of markers of sex-steroid enzymes in ovarian stroma\nTumor type\nAdjacent stroma a composite score b Distant stroma c composite score b\nP-value of adjacent\nvs distantd\nAKR1C3 CYP17 CYP19 HSD17 β1 Total e AKR1C3 CYP17 CYP19 HSD17 β1 Total\nEndometriosis-related\ntumors (atypical\nproliferative seromucinous\ntumor, endometrioid\ncarcinoma, clear cell\ncarcinoma, seromucinous\ncarcinoma) n =2 4\n7.88 6.5 1.8 0.22 4 3.75 0.5 0 0 1.13 0.0203\nLow-grade serous tumors\n(atypical proliferative\nserous tumor and low-grade\nserous carcinoma) n =2 5\n6 0.23 0.67 0.33 1.6 5.25 0 0 0 1.08 0.4251\nHigh-grade serous\ncarcinoma n =3 0\n6.25 1.72 1.31 0.96 2.54 8.79 0.52 0.52 1 2.56 0.9769\nMucinous tumors (atypical\nproliferative mucinous\ntumor, primary and\nmetastatic mucinous\ncarcinoma) n =1 5\n8.27 7.92 1.1 1 4.73 6.44 1.44 0.75 0.78 2.4 0.0167\naStromal cells immediately adjacent to epithelium. bAverage composite score (proportion × intensity). cStromal cells not immediately adjacent to\nepithelium (not present in all sections examined). dComparison of average total composite score in adjacent stroma and average total composite\nscore in distant stroma using t-test. eAverage composite score of all of the markers combined.\nFigure 3 Representative sections of endometriosis-related tumors with column a demonstrating an endometrioid carcinoma including a\nhematoxylin and eosin-stained (H&E) section. Strong nuclear immunoreactivity for progesterone receptor (PR) is seen in the malignant\nepithelium (similar expression of estrogen receptor (ER) and androgen receptor (AR) were also observed in this case; not illustrated). Sex-\nsteroid enzymes CYP17 and CYP19 are observed in the stroma immediately adjacent to the epithelium. Column b shows a clear cell\ncarcinoma including including a H&E-stained section. Nuclear immunoreactivity for steroidogenic factor 1 (SF-1) and cytoplasmic and\nnuclear immunoreactivity for calretinin are present in the stromal cells. Sex-steroid enzyme CYP17 is present in the stroma immediately\nadjacent to the epithelium.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\n568 LZ Blanco Jr et al\n\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\nLZ Blanco Jr et al 569\n\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\n570 LZ Blanco Jr et al\n\nmucinous tumors ( P o 0.0001) (Table 3). This was\nalso the case when individual markers were\ncompared.\nIn contrast, for low-grade serous tumors and high-\ngrade serous carcinoma, there was no statistically\nsignificant difference in the adjacent compared with\nthe distant stroma. However, in high-grade serous\ncarcinoma, calretinin was significantly higher in the\nadjacent vs the distant stroma ( P = 0.0289) while SF-1\nwas higher in the distant vs the adjacent stroma\n(P = 0.0279). No differences were observed in the\nindividual markers in low-grade serous tumors.\nMarkers of Sex-Steroid Enzymes ( CYP17, CYP19,\nHSD17b1, and AKR1C3)\nThere was significantly increased expression of these\ncombined markers in the adjacent compared with\nthe distant stroma for the endometriosis-related\ntumors ( P = 0.0203) and the mucinous tumors\n(P = 0.0167) (Table 4). More specifically, CYP17,a n\nenzyme involved in the biosynthesis of androgens,\nwas significantly higher in the adjacent compared\nwith the distant stroma in endometriosis-related\ntumors ( P = 0.0078) and mucinous tumors\n(P = 0.0010), but AKR1C3, another enzyme involved\nin androgen biosynthesis, was not significantly\nincreased in the adjacent compared with the distant\nstroma. Interestingly it was also expressed in the\nepithelium of the mucinous tumors. Expression of\nCYP19 (aromatase), which is involved in the con-\nversion of testosterone to estradiol, and HSD17β1,a n\nenzyme that converts low activity estrone to the\nmore potent estradiol, was not statistically different\nbetween the adjacent and distant stroma. There was\nalso no significant difference in the expression of\nthese markers in the stroma associated with low-\ngrade serous tumors and high-grade serous\ncarcinoma.\nMarkers of Sex-Steroid Hormone Receptors (Androgen\nReceptor, Estrogen Receptor, and Progesterone\nReceptor)\nExpression of ER was significantly higher in the\nepithelium compared with the adjacent stroma of\nendometriosis-related tumors ( P = 0.00001), low-\ngrade serous tumors ( P o 0.0001), and high-grade\nserous carcinoma ( P = 0.0036) (Table 5). Similarly,\nthere was a statistically significant increased expres-\nsion of ER in the epitheium compared with the\nadjacent stroma of low-grade serous tumors\n(P = 0.0003) and high-grade serous carcinoma\n(P o 0.0001) and PR in the epithelium compared\nwith the adjacent stroma of endometriosis-related\ntumors ( P = 0.0104), whereas there was significantly\ndecreased expression of ER ( P o 0.0001) and PR\n(P = 0.0108) in the epithelium compared with the\nadjacent stroma of mucinous tumors. In contrast, AR\nwas significantly decreased in the epithelium com-\npared with the adjacent stroma of endometriosis-\nrelated tumors and mucinous tumors.\nComparison of Sex-Steroid Enzyme Expression in\nStroma Immediately Adjacent to Epithelium and\nHormone Receptors in the Corresponding Epithelium\nand in the Stroma Immediately Adjacent to Epithelium\nOverall, 27 cases that expressed CYP17, which is\nessential for the biosynthesis of androgens, also had\nAR available for review. In endometriosis-related\ntumors, greater CYP17 expression correlated with\ngreater AR expression within the adjacent stroma in\nendometriosis-related tumors ( r\n2 = 0.8696,\nP = 0.0208, n = 5) and mucinous tumors ( r2 = 0.5366,\nP = 0.0104, n = 11). Interestingly, in high-grade serous\ncarcinoma ( r2 = 0.6630, P = 0.0486, n = 6), a statisti-\ncally significant inverse relationship was observed\nwith greater CYP17 expression in adjacent stroma\ncorrelating with less AR expression in the epithe-\nlium, suggesting a potential interplay between the\nenzyme and its corresponding hormone receptor.\nThese findings suggest that these enzymes may be\nproducing hormones that support the stroma itself in\nan autocrine fashion but not necessarily with the\nepithelium.\nA total of 44 cases with AKR1C3 expression also\nhad corresponding AR for analysis. Interestingly, in\nlow-grade serous tumors ( r\n2 = 0.5490, P = 0.0567,\nn = 7) there was a trend for increased AKR1C3\nexpression (an enzyme involved in androgen bio-\nsynthesis) in adjacent stroma with greater AR in\nepithelium, while in high-grade serous carcinoma,\n(r\n2 = 0.2509, P = 0.0176, n = 22), greater AKR1C3\nexpression significantly correlated with greater AR\nexpression within the adjacent stroma itself. These\nfindings also support a potential function of the\nstroma of producing androgens that stimulate the\nstroma as well as the epithelium.\nA total of 15 cases with CYP19, which is essential\nfor the biosynthesis of estrogens, also had corre-\nsponding ER for analysis. In high-grade serous\ncarcinoma, greater CYP19 significantly correlated\nwith greater ER expression within the adjacent\nFigure 4 Representative sections of serous tumors with column a showing a high-grade serous carcinoma including a hematoxylin and\neosin-stained (H&E) section. Strong nuclear immunoreactivity for estrogen receptor (ER) is seen in the malignant epithelium. Sex-steroid\nenzymes CYP17 and CYP19 are observed in the stroma immediately adjacent to the epithelium. Column b represents a low-grade serous\ncarcinoma including a H&E-stained section. Strong immunoreactivity for ER is seen in the epithelium, while focal cytoplasmic and\nnuclear immunoreactivity for calretinin is present in the stromal cells. Sex-steroid enzyme AKR1C3 is present in the stroma immediately\nadjacent to the epithelium.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\nLZ Blanco Jr et al 571\n\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\n572 LZ Blanco Jr et al\n\nstroma itself ( r2 = 0.7921, P = 0.0175, n = 6), again\nsuggesting a potential autocrine function in the\nadjacent stroma.\nNo other significant correlations were observed.\nExpression of Sex-Steroid Markers and Enzymes and\nHormone Receptors in Cortical Inclusion Cysts,\nOvarian Surface Epithelium, Endometriosis, and\nFallopian Tube\nExpression of calretinin, inhibin and SF-1 was\nsignificantly less in the adjacent compared with\nthe distant stroma for cortical inclusion cysts\n(P = 0.0479) and ovarian surface epithelium\n(P = 0.0002). The epithelium in cortical inclusion\ncysts had significantly greater hormone receptor\n(AR, ER, and PR) expression than the adjacent\nstroma ( P = 0.0262) but expression of sex-steroid\nenzymes in the adjacent stroma was low\nand did not differ from that of the distant stroma.\nStatistically significant differences were not\nobserved in ovarian surface epithelium with regards\nto these markers.\nThere was a trend of greater expression of sex-\nsteroid enzymes in the adjacent compared with the\ndistant stroma in endometriosis ( P = 0.0586) but\nFigure 5 Representative sections of mucinous tumors with column A demonstrating a mucinous carcinoma including a hematoxylin and\neosin-stained (H&E) section. Immunoreactivity for steroidogenic factor 1 (SF-1) is present in the stromal cells, while strong\nimmunoreactivity for progesterone receptor (PR) and androgen receptor (AR) is seen in the malignant epithelium. Sex-steroid enzymes\nCYP17 and CYP19 are observed in the stroma immediately adjacent to the epithelium. Column B shows an atypical proliferative\n(borderline) mucinous tumor including a H&E-stained section. Nuclear immunoreactivity for SF-1 and cytoplasmic immunoreactivity for\ninhibin are present in the stromal cells. Sex-steroid enzyme CYP17 is present in the stroma immediately adjacent to the epithelium.\nFigure 6 Representative sections of a cortical inclusion cyst with activated stroma including a hematoxylin and eosin-stained (H&E)\nsection. Cytoplasmic immunoreactivity for inhibin is present in the stromal cells. Sex-steroid enzyme AKR1C3 is focally present in the\nstroma immediately adjacent to the epithelium.\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\nLZ Blanco Jr et al 573\n\nthere were no significant differences in sex-steroid\nmarker and hormone receptor expression in these\nlesions.\nThe majority of both fallopian tube epithelium and\nadjacent stroma expressed ER, PR, AR, and AKR1C3,\nwhile in one-third of the cases adjacent stroma was\nimmunoreactive for calretinin. The other markers\nwere not expressed in either the epithelium or\nstroma. Overall, no significant findings were\nobserved in the fallopian tubes.\nDiscussion\nThe demonstration of enzymes involved in steroido-\ngenesis in the stroma immediately adjacent to\novarian tumors and the presence of corresponding\nhormone receptors in the epithelium of some of\nthese tumors confirms previous reports\n20,28,29 and\nprovides compelling evidence that the epithelium of\novarian tumors activates the surrounding stroma to\nproduce steroid hormones, and, that these hormones\ncan potentially stimulate the growth of the tumors. In\ncontrast to the typical ovarian stroma, but similar to\nthe theca interna surrounding developing follicles,\nthe ovarian stroma immediately adjacent to ovarian\nneoplasms is composed of lutein-like or theca-like\ncells which are particularly prominent in mucinous\ntumors, both primary and metastatic (Figure 2b).\nHowever, the overall number of mucinous tumors in\nthis series is small and therefore we are unable to\ndraw firm conclusions for this tumor type. Further,\ngiven that a majority of our cases were referred cases,\ncomplete clinical information including menstrual\nstatus were not available and could not be correlated\nwith our findings.\nSF-1 is a nuclear transcription factor that is\nthought to regulate the gene for the alpha subunit\nof inhibin and plays an important role in the\ndevelopment of the gonads and adrenal glands,\nsexual differentiation, reproduction, and\nmetabolism.\n30–33 In endometriosis-related and muci-\nnous tumors, SF-1 was expressed significantly more\nin the adjacent compared with the distant stroma.\nInhibin, a member of the transforming growth factor\nsuperfamily with growth promoting activities\n34 has a\nparacrine action in the ovary and regulates luteiniz-\ning hormone-induced androgen production by theca\ncells.\n35,36 We found that inhibin was significantly\nincreased in the stroma adjacent to mucinous\ntumors. Calretinin, a 29-kD calcium-binding protein\nstructurally related to inhibin,\n37 was also elevated in\nthe stroma adjacent to endometriosis-related, muci-\nnous tumors, and high-grade serous carcinoma.\nThe significantly increased expression of enzymes\ninvolved in the synthesis of sex-steroid hormones in\nthe stroma immediately adjacent to most of the\ntumors, especially endometriosis-related and muci-\nnous tumors, strongly suggests that locally produced\nsteroid hormones may support the growth and\nproliferation of these neoplasms. Of the four\nenzymes evaluated, CYP17, an enzyme that catalyzes\nthe C17 –C20 bond side chain cleavage of C21\nsteroids and is therefore essential for the biosynth-\nesis of androgens,\n38 was the most significantly\nexpressed enzyme in stroma of the various tumors,\nparticularly in endometriosis-related and mucinous\ntumors. More specifically, increased CYP17 corre-\nlated with increased AR expression in the stroma\nitself. Curiously, increased expression of CYP17 in\nthe stroma correlated with decreased expression of\nAR in the corresponding epithelium of high-grade\nserous carcinoma. These findings suggest an intricate\ninterplay between the neoplasms and the ovarian\nstroma, including a possible negative feedback\nmechanism between the hormones in the stroma\nand the receptors in the epithelium.\nIn attempting to understand the possible role of\nlocally synthesized steroid hormones and tumori-\ngenesis, the conversion of androgens to estrogen also\nneeds to be considered. A recent study suggests that\nsignificant amounts of estrogen are likely to be the\nresult of peripheral aromatization of androgens\n20 and\nalthough ovarian stromal cells express enzymes\nsupporting their capacity to convert androgens to\nestrogens,\n28,29 it remains to be seen whether this\noccurs locally. In our study no correlation was\nobserved between CYP17 in the stroma and ER in\nthe epithelium of any of the tumors. Similarly,\nAKR1C3, an enzyme that catalyzes the reversible\nconversion of the 17-keto and 17 β-hydroxy groups in\nandrogens and estrogens, including the conversion\nof androstenedione to testosterone,\n38,39 was consis-\ntently immunoreactive in the ovarian stroma of\nmany tumors in our study, but there was no\nsignificant correlation between AKR1C3 expression\nand AR expression in the neoplastic epithelium.\nConversely, increased AKR1C3 correlated with\nincreased AR in the adjacent stroma. This, along\nwith the CYP17 expression, suggests that androgens,\nin addition to being a source of estrogens, may be\nimportant in stimulating the stroma itself, acting in\nan autocrine fashion. Previous studies have impli-\ncated androgens in the pathogenesis of ovarian\ncancer\n40 and have reported that within the ovaries,\nthe secretion rate of androgens is higher than that of\nestrogens.41 In addition, animal models have indi-\ncated that testosterone stimulates the growth in vivo\nof ovarian surface papillomas and cystadenomas. 42\nCYP19 (cytochrome P450 aromatase, an enzyme\ninvolved in the final step of estrogen synthesis,\nconverting testosterone to estradiol\n17,38,43), was\nexpressed in several tumors, however no significant\nfindings were observed. Likewise, a significant\ncorrelation between CYP19 and ER in the neoplastic\nepithelium was not seen. However, there was a\ncorrelation with greater ER in the adjacent stroma\nitself, specifically in high-grade serous carcinoma,\nagain suggesting a possible autocrine function.\nHSD17β1, an enzyme that converts low activity\nestrone to the more potent estradiol,\n38 was the least\nfrequently expressed and no significant correlation\nModern Pathology (2017) 30, 563 –576\nSteroid hormone synthesis by ovarian stroma\n574 LZ Blanco Jr et al\n\nwas observed with ER expression in either the\nepithelium or in the stroma.\nOverall, our results showed that ovarian stroma,\nparticularly the stroma immediately adjacent to\nepithelium, was consistently immunoreactive for\ninhibin, calretinin and SF-1, CYP17, and AKR1C3,\nless frequently for CYP19, and rarely for HSD17β1.\nInhibin, calretinin and SF-1 identify hormonally\nactive cells whereas the enzymes specifically indi-\ncate that these cells are the site of steroid hormone\nsynthesis. The intense localization of these enzymes\nnot only in the stroma surrounding the tumor but\nalso within nests of tumor cells in contrast to the\nstroma distant from the tumors in the vast majority of\novarian neoplasms evaluated strongly suggests that\nthere is an interaction between the tumors and the\nstroma. Further, steroid hormone receptors, includ-\ning androgen receptor, were frequently expressed in\nthe epithelium of the vast majority of tumors with\nthe notable exception of mucinous tumors, therefore\ndemonstrating their capacity to be stimulated by the\nhormones produced by the stroma. The absence of\nhormone receptors in the epithelium of mucinous\ntumors may be due to their not being derived from\nmüllerian epithelium.\nIn endometriosis, activated ovarian stroma was\nalso present adjacent to epithelium. Although greater\ncomposite scores for expression of markers of\nsteroidogenesis and steroid enzymes were present\nin adjacent stroma in these lesions, the findings were\nnot statistically different compared with distant\nstroma. The finding that hormone receptors were\nconsistently expressed in endometriosis epithelium\nand adjacent stroma suggests that stroma may\nstimulate the epithelium in endometriosis as well.\nAnother interesting observation was the presence\nof activated ovarian stroma surrounding cortical\ninclusion cysts lined by tubal-type epithelium and\nthe presence of hormone receptors in the epithelium\nalbeit overall less than that of the ovarian tumors\n(Figure 6). We also evaluated ovarian surface\nepithelium and found minimal expression of SF-1,\nnegligible inhibin and only rare expression of only\nAKR1C3 in the underlying stroma, with some\nexpression of hormone receptors in the epithelium.\nDespite some immunoreactivity, the expression of\nmarkers of steroidogenesis and steroid enzymes was\nsignificantly less in the adjacent stroma surrounding\ncortical inclusion cysts and beneath the ovarian\nsurface epithelium compared with the distant ovar-\nian stroma. These findings suggest that unlike the\ntumors, benign epithelium does not lead to activa-\ntion of the stroma.\nIn conclusion, this study provides morphologic\nand immunohistochemical evidence that tumors in\nthe ovary activate the stroma to produce steroid\nhormones, most often androgens, and provides one\npossible explanation of why tumors thought to be\nderived from the fallopian tube and endometrium\nmay grow preferentially in the ovary. Additional\nstudies are needed to confirm whether the activated\novarian stroma surrounding neoplasms produces\nestrogens and androgens and, if so, whether their\nlevels are sufficient to stimulate and support tumor\ngrowth. The potential interaction of the stroma and\nthe ovarian carcinomas is highly provocative as it\nmay open the door for novel approaches to treatment\nas the activated ovarian stroma may serve as a\ntherapeutic target for anti-androgenic drugs.\nAcknowledgments\nWe would like to acknowledge Dr Howard Zacur,\nThe Theodore and Ingrid Baramky Professor of\nReproductive Endocrinology, Director of the Divi-\nsion of Reproductive Endocrinology and Infertility at\nThe Johns Hopkins Hospital, for his review of the\nmanuscript and helpful constructive criticisms.\nDisclosure/conflict of interest\nThe authors declare no conflict of interest.\nReferences\n1 Kurman RJ, Shih IeM. 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