{"paper_id":"fb2aa74b-66b9-4dc6-86e8-6ab95d1098b6","body_text":"Endometriosis, defined as the growth and development of endometrial-like tissue\n( i.e. , glands and stroma) outside the uterine cavity, is a gynecological\ndisease characterized by chronic pelvic pain, dyspareunia, dysmenorrhea, and infertility,\nthat affects 10 percent (%) of women of reproductive age [ 1 ]. The currently available treatments aim to decrease\nendometriotic lesion burden and to reduce inflammation and pain. Treatment options for\nendometriosis include non-hormonal [non-steroidal inflammatory drugs\n(NSAIDs)], and hormonal [gonadotropin-releasing hormone (GnRH), oral\ncontraceptives (OCs) and progestins] treatments [ 2 ]. However, inadequate responses and resistance to\ntreatment are common, and lead to symptom recurrence [ 2 ]. In addition, patients often report negative side effects that impair\ncompliance with these treatments [ 3 ].\nMany of these factors have contributed to the consensus that endometriosis has neither a\npharmacologic cure nor high therapy response rates, resulting in poor quality of life for\nwomen suffering from this incapacitating disease [ 4 – 6 ].\nThe pathophysiology of endometriosis is not well understood, but estrogen\ndependence and progesterone resistance are known to be important players in the\nestablishment and maintenance of endometriotic lesions [ 7 ]. The primary action of these ovarian steroid hormones is\nmediated through their cognate receptors, the estrogen receptors alpha and beta (ESR1 and\nESR2) and two progesterone receptor isoforms A and B (PGR-A and PGR-B) [ 8 ]. Previous studies have shown aberrant\nexpression of these hormone receptors in endometriosis lesions, including high ESR2 to ESR1\nratios and loss of expression of PGR [ 9 ]. These observations suggest that dysregulation in the signaling cascades\nmediated by these receptors and in the cellular behaviors they activate\n( e.g. , proliferation) are important factors not only in the etiology of\nendometriosis, but also in the observed inconsistency in therapeutic responses to the\nhormone treatments commonly used for this condition [ 10 ]. This variability in responses to treatment could be due to\ndifferences in the underlying pathophysiology of various disease presentations (e.g.,\novarian vs. peritoneal lesions) but also to differential expression of the drug’s\ntarget due to physiological factors or therapeutic stimuli [ 11 ]. Although it should be standard of care to ensure that\nall lesion types express the receptor being targeted (as in other hormonal conditions such\nas breast cancer), this is not currently taken into consideration in the treatment plan for\nendometriosis. Therefore, there is still a need to investigate the association between\nclinical responses and individual heterogeneity in the expression of steroid ovarian hormone\nreceptors in tissues that are biopsied during surgery for endometriosis. As a first step in\nthis process, we aimed to characterize the expression profile of ESR1, ESR2 and PGR in\ntissues from diverse lesion types based on their localization ( e.g. ,\novarian, peritoneal, fallopian, appendix, cecum, and skin & subcutaneous) as well as\nin eutopic endometrium (from patients and controls) representing the diversity of patients\ncommonly encountered in a clinical setting.\nThe present study was undertaken to expand and validate previous observations of\nhormonal receptor heterogeneity among endometriotic lesions and endometrial samples, taking\nadvantage of the availability of a Tissue Microarray (TMA) containing samples from\nendometriosis cases and controls. Immunohistochemical (IHC) analysis of this\nendometriosis-focused TMA allowed the identification of specific cells\n( e.g. , stroma, gland, endothelium, infiltrating inflammatory cells) and\ncellular localization ( e.g. , cytoplasmic vs. nuclear) in a large sample\nsize, and enabled direct comparisons while avoiding technical issues ( e.g. ,\ndecreasing variability on staining) [ 12 ]. We report here our characterization of different expression patterns of\nESR1, ESR2, PGR, and Ki67 (a marker of heightened cellular proliferation) in eutopic and\nectopic endometrium, and discuss the potential implications for the clinical management of\nindividual patients with endometriosis.\n\nUsing protocols approved by our institution IRB committee (IRB #050207,\nIF), a total of 164 cores from 83 de-identified archived formalin fixed paraffin-embedded\nendometrium and endometriosis tissue blocks were obtained from a Pathology Laboratory from\nSouthern Puerto Rico between the years of 2000-2009. Tissues were evaluated by two\npathologists (MG and AM) to confirm the diagnosis of endometriosis, to select areas of the\nblock where endometriosis (defined as glands and stroma) was present, and to determine the\nmenstrual cycle phase of the endometrial samples. There were 5 cases of matched samples\n(lesion and endometrium); only for those we could determine menstrual cycle phase\n(proliferative n=2; secretory n=3). The blocks were used to construct a\ntissue microarray (TMA) at the Moffitt Cancer Center (Moffitt) Tissue Core that has\npreviously been described and used for various IHC studies [ 13 – 15 ].\nDuplicate core biopsies in the TMA came from the following tissue types: endometriosis\nlesions localized in the ovaries (n=29), fallopian tubes (n=16),\nperitoneum (n=34), skin (umbilical region) (n=4), and gastrointestinal\ntract [cecum and appendix] (n=7). Due to lower number of tissue\nsamples in the skin (n=4), appendix (n=4) and cecum (n=3) groups,\nthese were pooled for the analysis and categorized as extra-pelvic endometriosis.\nAdditionally, the TMA included eutopic endometrial samples from women with endometriosis\nand controls in either proliferative or secretory phases. Control endometrial samples were\nobtained from women undergoing hysterectomy for benign gynecological conditions such as\nuterine fibroids of dysfunctional bleeding, who had normally cycling endometrium (not\nhyperplastic, not menopausal, in either proliferative or secretory phase) as per\npathological analysis. The mean age of the endometriosis cases was 40.0 ±7.0 years\n(endometrial samples) and 35.8 ± 8.7 years (lesions) compared to 46.1 ±\n7.7 of controls, which was not significantly different.\nImmunostaining for ESR1, ESR2, PGR, and Ki67 was conducted at Moffitt’s\nTissue Core following standardized protocols. Antibody dilutions and experimental\nconditions were determined after standardization and normalization procedures prior to the\nexperiments. Individual TMA 10 μm slides were incubated with the following primary\nantibodies: ESR1 (Genentech, cat# GTX29269), ESR2 (Genentech, cat#\nGTX70174), PGR (Abcam, cat# ab131486) and Ki67 (Novus, cat# NB110-57147).\nDifferences between PGR-A and PGR-B were not assessed because the antibody employed for\nthe staining does not discriminate between isoforms. Thus, nuclear immunostaining for PGR\nis reported as global PGR expression. The slides were scanned at Moffitt’s\nAnalytical Microscopy Core using the Aperio Digital Pathology Scanner (Buffalo Grove, IL).\nThe Image Scope Software (Aperio) was used to select areas representative of glands and\nstroma of the tissues in the TMA to be analyzed digitally for nuclear immunostaining\nintensity of ESR1, ESR2, PGR, and Ki67. Data were analyzed as percent (%) of\npositive nuclei presented as average per tissue type (glands vs stroma).\nStatistical analysis included non-parametric Mann Whitney test to analyze\ndifferences in age among groups and non-parametric ANOVA (Kruskal-Wallis test) followed by\na Dunn’s Post-Hoc test to analyze the significance of differences in the\npercentage of positive nuclei for the hormone receptors and Ki67 among the groups (lesion\ntype, proliferative and secretory endometrium from cases and controls). All the\nstatistical analyses were performed using SPSS v20 and GraphPad Prism was used for\ngraphical representation of data. Statistical significance was set at p<0.05.\n\nOvarian lesions showed the lowest glandular ESR1 expression, which was\nsignificant compared to fallopian (p<0.001), peritoneal (p<0.05), and\nextra-pelvic lesions (p<0.01). ( Figure 1 \npanel A). In the stromal compartment of the lesions, we observed similar results with\novarian lesions showing the lowest level of ESR1 expression, significant when compared to\nfallopian tube (p<0.001) and extrapelvic lesions (p<0.001) ( Figure 1  panel B). In stroma of lesions, 10.8% of the\nsamples show loss or very low (>10%) ESR1 positivity (16 out of 83).\nRegarding endometrial tissues, significant differences were observed in ESR1\nnuclear expression levels between the stroma of proliferative endometrium from patients\nand controls (p <0.05) ( Figure 1  panels C and\nD). Also, we observed a high number of secretory endometrium samples from patients with\nloss or very low (>10%) ESR1 positivity in glands (45.5%; 10/22)\nand stroma (45%; 9/20) compared to controls (glands: 10.5%, 2/19; stroma:\n26.3%, 5/19).\nWe observed high levels of ESR2 nuclear positivity in the glandular compartment\nof all lesions. The highest percentage of positive nuclei was seen in the ovarian and\nfallopian tube lesions (significantly different from peritoneal and extra-pelvic lesions)\n( Figure 2  panel A). In stroma, the only significant\ndifference observed in ESR2 protein levels was between extra-pelvic and fallopian tube\nlesions ( Figure 2  panel B).\nIn the glandular compartment of the endometrial tissues, secretory endometrium\nfrom controls expressed the highest percentage of ESR2 positive nuclei, which was\nsignificantly different from secretory endometrium from patients (p<0.01) ( Figure 2  panel C). Statistical significance was also\nobserved between secretory and proliferative endometrium from controls (p<0.0001);\nhowever, no significant differences were observed when comparing proliferative and\nsecretory endometrium from patients ( Figure 2  panel\nC). Although the percentage of ESR2 positivity was lower in proliferative endometrium from\ncontrols compared to patients in both glands and stroma, these differences did not reach\nstatistical significance ( Figure 2  panel D).\nIn glands, the highest percentage of nuclear positivity for PGR was observed in\nextra-pelvic endometriotic lesions, followed by fallopian tube and peritoneal lesions.\nOvarian lesions were characterized by a wide range of PGR positivity, from 99% to\n3% ( Figure 3  panel A). Significant\ndifferences were observed between extra-pelvic lesions compared to ovarian\n(p<0.001). Loss of PGR expression was most striking in ovarian lesions, which had\nthe highest proportion of lesions with null or very low (>10%) PGR nuclear\npositivity in both glands (56.3%; 9/16) and stroma (68.8%; 11/16) ( Figure 3  panels A and B).\nExpression of PGR in glands was higher in the proliferative endometrium of both\npatients and controls compared to secretory endometrium; however, endometrium from\npatients had a higher proportion of null or very low (>10%) PGR expression\nin glands (33.3; 5/15) and stroma (28.6%; 4/14), and a broader range of PGR\npositivity (glands: 97-51%; stroma: 94 to 35%). ( Figure 3  panels C and D). No differences were observed in PGR\nexpression in stroma of the endometrial samples ( Figure\n3  panel D).\nWe next calculated the ratio of percent positive nuclei in ESR2 compared to ESR1\nin all lesions and endometrial samples in the TMA ( Figure\n4 ). The largest ESR2 to ESR1 ratio was observed in ovarian lesions compared to\nperitoneal (p<0.001), fallopian tube (p<0.001), and extra-pelvic lesions\n(p<0.0001) in glands, and compared only to fallopian tube (p<0.0001) in\nstroma ( Figure 4  panels A and B). ESR2 to ESR1 ratios\nwere highest in secretory vs. proliferative endometrium from both patients and controls in\nglands and stroma ( Figure 4  panel C and D). No\nsignificant differences were observed when comparing endometria obtained at the same\nmenstrual cycle phase from patients and controls.\nFive cases had matched eutopic and ectopic endometrium samples in the TMA. For\nthose we were able to compare the pattern of receptor expression. Although the number of\ncases is small, analysis of samples from matched ectopic and eutopic endometrium uncovered\ninteresting trends ( Figure 5 ). Ovarian endometriosis\ncases (1 and 2) were characterized by higher levels of ESR2 vs. the other receptors\nregardless of menstrual phase. The most striking difference between the two cases of\novarian endometriosis was the negligible ESR1 expression in eutopic endometrium seen only\nin the patient at the secretory phase, which was not seen in the two other cases at\nsecretory phase (case 3 with fallopian tube and case 5 with peritoneal lesions). Fallopian\nendometriosis cases (3 and 4) differed only on the higher positivity for ESR1 seen in\nproliferative endometrium. Peritoneal endometriosis (5) showed a tendency for lower levels\nof ESR1 than the other receptors that can be attributed to the menstrual phase\n(secretory). One case (6) with multiple lesions showed low PGR expression only in ovarian\nlesions (n.s.).\nThe percentage of Ki67 positive nuclei was analyzed based on cutoffs commonly\nused in determining cancer prognosis [favorable if staining <10%\n(low), borderline from 10% to 20%, and unfavorable if staining\n>20% (high)] [ 16 ]. We observed that extra-pelvic (75%) and fallopian tube\n(54%) lesions had the highest (>50%) proportion of samples with\n>10% Ki67 positivity ( Figure 6 ).\nDifferences in Ki67 positivity were statistically significant only in glands of\nextra-pelvic vs. ovarian and vs. peritoneal lesions (p<0.05). In glands of the\nendometrial tissues, the secretory endometrium from patients had a higher proportion of\nsamples with >10% Ki67 positive nuclei compared to the secretory\nendometrium from controls (60% vs. 15%).\n\nAberrant expression and signaling of ESR1, ESR2 and PGR have been associated with\nthe development and progression of endometriosis, which is commonly referred to as\nestrogen-dependent and progesterone-resistant [ 17 ]. Consequently, most treatments used today for endometriosis target\nthese nuclear receptors by either blocking their action or promoting signaling actions.\nThus, immunohistochemical characterization of the nuclear expression of ESR isoforms and PGR\nin target tissues that are biopsied during surgery could help determine response to commonly\nprescribed hormonal treatments. This is standard of care in other hormonal conditions such\nas breast cancer. However, the association between therapeutic responses, recurrence rates,\nand expression of ESR1/2 and PGR in endometriosis has not been well characterized. Using IHC\nand automated image analysis of percentage of positive nuclei, we observed significant\ndifferences in the pattern of hormone expression based on lesion localization and also by\nendometrial cell compartment ( e.g. , glands vs. stroma). Ovarian lesions\nshowed the lowest expression of ESR1 and PGR in both glands and stroma, and the highest\nexpression of ESR2 in glands only. All ovarian implants had 100% of ESR2 nuclear\nimmunostaining in glands. Fallopian tube lesions in general showed high expression of ESR1/2\nand PGR. Extra-pelvic endometriotic lesions showed the highest ESR1 and PGR expression and\nthe lowest ESR2 expression (only in glands). The most striking differences among endometria\nwere observed for ESR2 positivity, which was highest in the glandular epithelium of both\nproliferative and secretory endometrium from patients. We also observed a range in the\nlevels of PGR in the glands and stroma of proliferative endometria from cases, compared to\ncontrols that showed much less variability in the staining; however, the physiological\nimplications of these differences are still unknown. Although endometrium from patients and\ncontrols as well as endometriotic lesions have been shown to differ in their capability of\nresponding to P4 due to low level expression of the PGR isoforms A and B [ 18 ], this study showed that not all lesion types\nare characterized by loss of PGR expression (one example being extra-pelvic lesions).\nTherefore, ‘P4 resistance’ may not be a universal characteristic of the\ndisease in its varying manifestations and across all patients at a given time depending on\ntheir lifetime history of disease. Whether the observed heterogeneity in hormone receptor\nexpression in different types of tissues could explain differences in patient responses to\nhormonal treatments still needs to be evaluated.\nIt has been speculated that high levels of ESR2 in endometriotic tissues\nsuppresses ESR1 and PGR expression, thus contributing to increased proliferation in response\nto E2, as well as to P4 resistance [ 19 ,  20 ]. Therefore, we next evaluated the ESR2 to\nESR1 ratios of nuclear positivity in all tissues in the TMA. We show here that some, but not\nall, endometriosis lesion types are characterized by high ESR2:ESR1. Ovarian endometriotic\nlesions in particular had high levels of ESR2 positivity compared to ESR1 in both glands and\nstroma. Higher ESR2 to ESR1 ratios were also observed in secretory endometria from both\npatients and controls. However, no significant differences in the ESR2 to ESR1 ratio were\nobserved in the proliferative phase endometrial tissues. The majority of published studies\nhave shown overexpression of ESR2 relative to ESR1 in tissues from endometriosis patients\n[ 9 ,  20 ,  21 ], except two studies showing\nthat 1) both eutopic endometrium from patients and ovarian endometriosis had predominantly\nhigher levels of ESR1 than ESR2 at the mRNA level [ 22 ] and 2) endometriotic stromal cells derived from ovarian chocolate\ncysts expressed the same levels of ESR1 and ESR2 mRNA [ 23 ]. These studies suggest that ESR1 and ESR2 may\ncontribute independently to the estrogenic cues that trigger specific cellular actions or\nthat estrogen receptor heterodimerization could lead to a state of co-dependency that\nmediates the estrogen-induced phenotype observed in endometriosis. In hormone-dependent\nmalignancies such as breast, prostate, and endometrial cancers, skewed ESR2:ESR1 have been\ncorrelated with tumor grade and clinical outcomes including overall survival rates\n[ 24 ,  25 ]; however, clinical correlations of ESR2:ESR1 in endometriosis still\nneed to be conducted that should take in consideration the type of lesions present.\nImmunohistochemical assessment of Ki67 positive cells has been widely employed to\nassess the cellular proliferation capacity in benign and malignant diseases [ 26 ,  27 ],\nand as a biomarker of poor prognosis, lower survival, and therapy resistance in cancer\n[ 28 ,  29 ]. Clinically, if >10% of nuclei are positive the cancer\nis considered to have borderline to unfavorable prognosis [ 30 ]. Meanwhile, data regarding Ki67 expression in eutopic\nand ectopic endometrium are contradictory [ 31 ,  32 ], although it is generally\naccepted that Ki67 positivity is higher in eutopic proliferative endometrium than in\nendometriotic lesions [ 33 ,  34 ]. In this study, we observed that Ki67 nuclear\npositivity of over >10% was most prominent in glands of fallopian tube and\nextra-pelvic lesions, as well as in secretory endometrium from patients compared to the same\ntissues from controls. As expected, we also observed higher levels of Ki67 nuclear staining\nin proliferative endometrium from both cases and controls. In view of the fact that a\ndiagnosis of endometriosis has been associated with increased risk of cancer (endometrioid\nand clear cell subtypes) [ 35 ], and\nthat carcinogenic transformation of fallopian tube epithelium is associated to ovarian\ncancer [ 36 ], it would be important to\nassess the Ki67 index of lesions that are surgically removed in order to have a better\nunderstanding of the clinical picture and prognosis of individual patients.\nThere are few studies using TMAs to elucidate hormone receptor status of\nendometriosis [ 37 – 39 ]. To our knowledge, this is the first study using a TMA\nthat includes pelvic endometriotic lesions (ovarian, peritoneal, and fallopian tube),\nextra-pelvic endometriotic lesions (skin & subcutaneous, cecum and appendix), as\nwell as endometria from patients and controls to evaluate hormone receptor expression. Our\nresults highlight 1) the value of the TMA technology to screen for potential biomarkers with\ndiagnostic, prognostic, and therapeutic value (while controlling for potential experimental\nvariables), and 2) the importance of analyzing different lesion types. Side by side analysis\nof hormone receptor expression in matched eutopic and ectopic endometrium uncovered\ninteresting trends and lesion-specific differences in hormone receptor expression. For\ninstance, ovarian endometriosis was the only tissue type showing predominant expression of\nESR2 regardless of menstrual phase. In contrast, ESR1 levels were lower in ovarian\nendometriosis compared to fallopian tube, although both patients were in the proliferative\nphase. And ESR1 levels were almost negligible only in secretory endometrium from patients\nwith ovarian endometriosis, not in those with fallopian or peritoneal disease. Evidently,\nthese results need to be validated in larger studies.\nOur study is limited by the nature of the TMA as it was constructed with archived\nlesions and endometrium biopsies obtained in a de-identified fashion. Therefore, there is no\ninformation on previous treatments and natural history of the disease. Also, while some\ncases had matched lesions and eutopic endometrium allowing for important comparisons, for\nothers information about menstrual cycle phase was lacking. However, apart from known\nchanges in ESR1 level between phases, no apparent differences were observed in ESR2 and PGR\nexpression in lesions from which menstrual cycle phase was known. Our study is also limited\nin that it does not take into account differences in expression of ESR1 in mid-late luteal\nvs. late follicular phase, of PGR levels between early and late proliferative phase, or\nbetween PGR-A and PGR-B in early vs late secretory phase, for which significant differences\nhave been reported [ 40 ,  41 ]. Despite these limitations, this study is valuable as\nit includes samples from patients with diverse clinical and treatment histories, as would\ntypically be encountered in a clinical setting. Other strengths of our study include the\ncharacterization of receptor positivity by glands and stroma, and the use of an automated\nimaging analysis software that reduces the variability introduced by semi-quantitative\nanalysis by independent scorers.\nThe differences in hormone receptor expression levels among lesions shown here and\nby others may be indicative of 1) different pathophysiological mechanisms underlying their\ndevelopment [ 42 ], ii) menstrual cycle\nphase [ 41 ], and iii) different\nlifetime history of hormonal and other treatments. For instance, it is well known that ESR1\nlevels will significantly decrease after prolonged exogenous therapy with\nprogestins[ 43 ]. Regardless of the\nunderlying reason, these differences could explain the observed variability in therapeutic\nresponses to hormonal treatments among patients. The TMA used in this study includes a wide\nvariety of subjects, likely to have variable lifetime experiences, treatments, exposures and\nclinical histories, akin to what a community gynecologist will have to treat in his/her\ngeneral practice. Moving forward towards a personalized approach to patient management using\nhormonal treatments, heterogeneity in expression of ovarian steroid hormone receptor\nexpression is not only expected, but imperative to test in order to customize the treatment.\nThe wider the variability observed (refer to ESR1 levels in ovarian lesions vs. fallopian\ntube in  Figure 1A ) the greater the need to test the\nsample before prescribing any hormonal drug because wide variations in receptor expression\nare to be expected from one patient to the next. Also quite telling is to see surprisingly\nlow variability across patients for ESR2 levels in glands seen in  Figure 2 , despite subjects likely having heterogeneous treatments\nand exposures. Whether this can be considered a tell-sign of endometriosis would need to be\nconfirmed in larger studies.\nThe results of this study have high translational value as they suggest that a\ngynecologist would be able to select the most appropriate hormonal treatment\n[ e.g. , oral contraceptives, oral or IUD progestins] based\non the particular pattern of expression of the ESR1, ESR2 and PGR of individual\nlesions that are surgically resected and characterized by IHC in a given\npatient. While these data could also suggest the potential use of selective\n[ 44 ](SERMs, e.g., raloxifene) and\nselective progesterone receptor modulators (SPERMs, e.g., mifepristone, asoprisnil),\nsynthetic steroidal drugs that act as agonists or antagonists of ESR1/2 or PGR,\nrespectively, it is important to note that these drugs have not yet been approved for the\ntreatment of endometriosis [ 3 ,  44 – 46 ]. The\nresults of this study would require of further clinical validation before widespread\napplication, and follow-up studies to correlate receptor expression with previous treatments\nand clinical responses are warranted. Our data support the need to implement personalized\nmedicine strategies in endometriosis that take in consideration biological differences of\nthe lesions and their clinical implications.","source_license":"CC0","license_restricted":false}