{"paper_id":"0476ae64-840a-4825-a72f-3b8adc1e1c56","body_text":"Aromatase expression in low-grade\nendometrial stromal sarcomas: an\nimmunohistochemical study\nOlaf Reich 1 and Sigrid Regauer 2\n1Department of Obstetrics and Gynecology and 2Department of Pathology, University of Graz, Graz, Austria\nAromatase expression has been described in stromal cells of endometriosis, adenomyosis and endometrial\ncancer. We analyzed aromatase expression in a series of 23 low-grade endometrial stromal sarcomas. Archival\nformalin-fixed and paraffin-embedded material was analyzed with immunohistochemistry. Aromatase expres-\nsion was evaluated with a monoclonal and a polyclonal antibody using the peroxidase–antiperoxidase method.\nA score was calculated based on the percentage of positive tumor cells and the staining intensity. Aromatase\nwas seen in 19 (83%) of 23 tumors with monoclonal antibody and 20 (87%) of 23 tumors with polyclonal\nantibody. Aromatase expression using the monoclonal antibody was scored as high in five (22%), moderate in\nnine (39%) and low in five (22%) tumors. Four (17%) low-grade endometrial stromal sarcomas did not stain for\naromatase. Aromatase expression with the polyclonal antibody was scored as high in seven (31%), moderate in\nfour (17%) and low in nine (39%) tumors. Three (13%) low-grade endometrial stromal sarcomas did not stain for\naromatase. Little or no aromatase expression tended to correlate with stage I disease, while higher scores were\nmore frequently associated with advanced disease. Our results demonstrate that most low-grade endometrial\nstromal sarcomas express aromatase. The staining pattern, however, is heterogeneous. The high percentage of\naromatase positivity in low-grade endometrial stromal sarcomas may have implications in the management of\nthese tumors and offer new treatment modalities such as hormonal therapy with aromatase inhibitors.\nModern Pathology (2004) 17, 104–108, advance online publication, 19 November 2003; doi:10.1038/modpathol.3800031\nKeywords: endometrium; stromal sarcoma; aromatase; treatment\nLow-grade endometrial stromal sarcomas are rare,\naccounting for about 0.2% of all genital tract\nmalignancies.1 They occur predominantly in pre-\nmenopausal women. 2 Little is known of their\netiology or natural history. Low-grade endometrial\nstromal sarcomas are hormone sensitive and estro-\ngen can act as a growth stimulus. 3 Most women with\nlow-grade endometrial stromal sarcomas undergo\nbilateral salpingo-oophorectomy as part of primary\ntreatment. However, estrogen can also be produced\nby extraovarian sources and likely by tumor cells\nthemselves.4–7 This extraovarian estrogen produc-\ntion depends on conversion of circulating androgen\nto estrogen via the aromatase enzyme complex. The\naromatase enzyme complex consists of the CYP19\ngene product aromatase cytochrome P450 and a\nflavoprotein NADPH-cytochrome P450 reductase\nand is located in the endoplasmic reticulum of\nestrogen-producing cells. 4\nTseng et al 8 reported concentrations of aromatase\nin eight uterine sarcomas, which were within the\nrange of activity found in normal proliferative\nendometrium or above normal levels. These authors\nused cytosol-based biochemical examinations and\ntheir observations have not been confirmed in larger\nstudies. Immunohistochemical quantification of\naromatase has not been reported for low-grade\nendometrial stromal sarcomas. In the present study,\nwe analyzed aromatase expression in a series of 23\nlow-grade endometrial stromal sarcomas using\nmonoclonal and polyclonal antibodies.\nMaterials and methods\nHistologic sections of 23 low-grade endometrial\nstromal sarcomas treated between 1983 and 2002\nwere retrieved from the archives and re-evaluated by\ntwo pathologists (SR and OR). All tumors were\ncomposed of cells resembling normal endometrial\nstromal cells invading the myometrium and vascular\nspaces. Mitoses were fewer than 10/10 high-power\nReceived 16 June 2003; revised 12 August 2003; accepted 18\nAugust 2003; published online 19 November 2003\nCorrespondence: O Reich, Department of Obstetrics and Gyneco-\nlogy, University of Graz, Auenbruggerplatz 14, A-8036 Graz,\nAustria.\nE-mail: olaf.reich@kfunigraz.ac.at\nModern Pathology (2004) 17, 104–108\n& 2004 USCAP , Inc All rights reserved 0893-3952/04 $25.00\nwww.modernpathology .org\n\nfields (HPF ¼ objective /C240) and necrosis was\nabsent. The tumors were staged according to\nthe International Federation of Gynecology and\nObstetrics (FIGO) classification for endometrial\ncarcinoma.9\nFor immunohistochemical analysis, one or two\nparaffin blocks with representative portions of the\ntumors were selected and 2- mm thick sections were\nstained with monoclonal (1:100 dilution, SM1671P ,\nAcris, Germany) and polyclonal antibody (1:1000\ndilution). The anti-aromatase polyclonal antibody\nwas the rabbit immunoglobulin fraction of an\nantiserum prepared against human placental aroma-\ntase as previously decribed 10 and was kindly\nprovided by Dr N Harada, Fujita Health University\nHospital of (Toyaoake, Japan). The peroxidase–\nantiperoxidase method was used to detect the\nantigens. Aromatase expression was evaluated by\ntwo pathologists (SR and OR). Placental tissue\nserved as positive controls. A score was calculated\nbased of the percentage of positive tumor cells\n(o10% ¼ 1; 11–50% ¼ 2; 51–80% ¼ 3; 481% ¼ 4)\nmultiplied by the staining intensity (negative ¼ 0;\nweak ¼ 1; moderate ¼ 2; strong ¼ 3). A minimum of\n1% of tumor cells had to be positive to be called\npositive staining. A final score of 0 was classified as\nnegative, 1–3 as low; 4–6 as medium and 8–12 as\nhigh.\nThe mean age of the patients at surgery was 45\nyears (range 25–79) and only four patients were\npostmenopausal. A total of 15 patients had surgical\nstage I, seven had stage III, and one stage IV disease.\nIn total, 22 patients underwent total abdominal\nhysterectomy with bilateral salpingo-oophorectomy.\nOne patient (pt.) had only exploratory laparotomy\n(pt. 5). Seven patients (pts. 1–4, 7, 8, 23) with stage\nIII disease additionally had an omentectomy and\none patient (pt. 7) underwent pelvic lymphadenec-\ntomy. Six patients (pts. 1, 3, 4, 7, 9, 23) recieved\nadjuvant chemotherapy, one patient (pt. 2) adjuvant\nradiation, and 16 patients no further treatment.\nSeven patients were lost to follow-up and the\nmedian follow-up of the remaining 16 patients was\n8.2 (1–16) years. Five patients (pts. 3, 4, 7, 9, 17)\ndeveloped pelvic recurrences and one patient (pt. 5)\ndeveloped lung and bone metastases. T wo patients\n(pts. 2 and 5) died of disease and one patient (pt. 18)\nof unrelated causes.\nResults\nAromatase was seen in 19 (83%) of 23 tumors with\nmonoclonal antibody and 20 (87%) of 23 tumors\nwith polyclonal antibody. Aromatase expression\nusing the monoclonal antibody was scored as high\nin five (22%), moderate in nine (39%) and low in\nfive (22%) tumors (Figures 1 and 2). Four (17%)\nlow-grade endometrial stromal sarcomas did not\nstain for aromatase. Aromatase expression with the\npolyclonal antibody was scored as high in seven\n(31%), moderate in four (17%) and low in nine\n(39%) tumors. Three (13%) low-grade endometrial\nstromal sarcomas did not stain for aromatase\n(Table 1).\nLow-grade endometrial stromal sarcomas with a\nhigh score for aromatase were uniformly positive\nthroughout the entire tumor with strong and diffuse\ncytoplasmic staining (Figure 1). Low-grade endome-\ntrial stromal sarcomas with a moderate score\nshowed variable staining. In low-grade endometrial\nstromal sarcomas with a low score, the majority of\nthe tumor cells were negative. The staining intensity\nof positive cells ranged from weakly to strongly\npositive with either a diffuse or a granular cytoplas-\nmic staining pattern (Figure 2).\nDespite the small study numbers, low-grade\nendometrial stromal sarcomas with no or low\naromatase expression tended to correlate with stage\nI disease, while higher scores were more frequently\nFigure 1 Low-grade endometrial stromal sarcoma with overall\nhigh aromatase expression with homogeneous strong diffuse\ncytoplasmic staining in the absence of nuclear staining (pt. 4,\nscore 12). Antiaromatase, monoclonal, peroxidase–antiperoxi-\ndase, original magnification /C2200.\nFigure 2 Low-grade endometrial stromal sarcoma with overall\nlow aromatase expression. Only individual cells or small groups\nof cells show strong granular cytoplasmic staining, while the\nmajority of cells do not stain specifically (pt. 14, score 3).\nAntiaromatase, monoclonal, peroxidase–antiperoxidase, original\nmagnification /C2200.\nAromatase in endometrial stromal sarcoma\nO Reich and S Regauer\n105\nModern Pathology (2004) 17, 104–108\n\nassociated with advanced stages (Table 1). In the\naromatase-positive group, 10 of 12 patients with\nstage I disease had low or medium scores, only two\npatients (pts. 13 and 20) had a high score. With the\nexception of patient 23, all aromatase-negative low-\ngrade endometrial stromal sarcomas corresponded\nto stage I disease (pts. 10–12). Of the five patients\nwith recurrent disease (pts. 3, 4, 7, 9, 17) one had a\nlow score (pt. 9), two had a medium score (pts. 3 and\n17), and two had a high score (pts. 4 and 7). The two\nwomen who died of disease (pts. 2 and 5) had a\nscore of 8 (high) and 12 (high), respectively (mono-\nclonal or polyclonal antibody, Table 1).\nDiscussion\nAromatase expression has been described in stromal\ncells of endometriosis, adenomyosis and endome-\ntrial cancer,4,11–13 but not in stromal cells of eutopic\nendometrium or endometrial hyperplasia. 13 This is\nthe first immunohistochemical study of aromatase\nexpression in low-grade endometrial stromal sarco-\nmas. We found that most tumors expressed aroma-\ntase (Table 1 and Figures 1 and 2). There was a wide\nagreement among the staining results obtained with\nmonoclonal and polyclonal antibody, and the\ndifferences were limited to one category (Table 1).\nStaining was heterogeneous. The small number of\npatients in our study precludes speculation about\nassociations between clinical behavior and this\nheterogeneous aromatase expression, but low scores\nappeared to be more common in stage I disease,\nwhile higher scores were more frequently seen in\npatients with advanced diseases. The two women\nwho died of disease showed high aromatase expres-\nsion (Table 1).\nAromatase is a key enzyme in estrogen biosyn-\nthesis, and estrogen seems to be a promoting factor\nin low-grade endometrial stromal sarcomas. 3 Low-\ngrade endometrial stromal sarcomas have been\nreported in women with exogenous or endogenous\nhyperestrogenism.14–16 Among women with stage I\nlow-grade endometrial stromal sarcomas, a higher\nrecurrence rate is found in patients with residual\novarian tissue when compared with those treated\nwith bilateral oophorectomy. 14 Similarly, regression\nin size of metastases have been reported after\nbilateral oophorectomy 17 and radiation-induced\ncastration.18 A lower incidence of recurrence and\nlonger disease-free survivals after bilateral oopho-\nrectomy have also been reported. 19\nRecurrences develop in one-third to one-half of\npatients with low-grade endometrial stromal sarco-\nmas and can appear as long as 30 years after primary\ntherapy.20–22 Little is known about the reasons for\nsuch a high recurrence rate after primary surgery. A\npossible explanation could be a growth stimulus by\nestrogen on residual tumor cells. After oophorec-\ntomy, estrogen can be produced by peripheral\nTable 1 Expression of aromatase in 23 patients with low-grade endometrial stromal sarcomas\nPt.\nno.\nAge\n(years)\nStage\n(FIGO) at\npresentation\nAdjuvant\ntherapy\nClinical\nfollow-up\nAnti-aromatase\nmonoclonal\nInterpretation\n(overall\nexpression)\nAnti-aromatase\npolyclonal\nInterpretation\n(overall\nexpression)\nCells SI Score Cells SI Score\n1 50 III CT Lost 3 3 9 High 4 2 8 High\n2 65 III RT DOD 4 3 12 High 3 3 9 High\n3 44 III CT AWD 3 2 6 Medium 2 3 6 Medium\n4 35 III CT AWD 4 3 12 High 4 3 12 High\n5 35 IV — DOD 3 2 6 Medium 4 2 8 High\n6 46 I — NED 1 2 2 Low 1 3 3 Low\n7 35 III CT AWD 4 2 8 High 3 3 9 High\n8 38 III — NED 2 1 2 Low 1 3 3 Low\n9 35 I CT AWD 1 2 2 Low 1 1 1 Low\n10 47 I — NED 0 0 0 Negative 0 0 0 Negative\n11 74 I — Lost 0 0 0 Negative 0 0 0 Negative\n12 34 I — NED 0 0 0 Negative 0 0 0 Negative\n13 38 I — NED 3 3 9 High 3 3 9 High\n14 50 I — NED 1 3 3 Low 1 3 3 Low\n15 45 I — NED 2 3 6 Medium 1 3 3 Low\n16 25 I — Lost 1 3 3 Low 1 2 2 Low\n17 35 I — AWD 2 2 4 Medium 1 2 2 Low\n18 79 I — DWD 2 2 4 Medium 2 2 4 Medium\n19 39 I — Lost 2 3 6 Medium 2 2 4 Medium\n20 34 I — Lost 2 2 4 Medium 3 3 9 High\n21 72 I — Lost 3 2 6 Medium 3 1 3 Low\n22 44 I — NED 2 2 4 Medium 2 2 4 Medium\n23 42 III CT Lost 0 0 0 Negative 1 1 1 Low\nCT, chemotherapy; RT, radiotherapy; NED, no evidence of disease; AWD, alive with recurrent disease; DWD, dead without disease; DOD, dead of\ndisease; Lost, lost to follow-up. Cells: Percentage of positive tumor cells ( o10% ¼ 1; 11–50% ¼ 2; 51–80% ¼ 3; 481% ¼ 4). SI: Staining intensity\n(negative ¼ 0; weak ¼ 1; moderate ¼ 2; strong ¼ 3). Score: Cells /C2SI, Interpretation: Score 1–3 as low; 4–6 as medium and 8–12 as high.\nAromatase in endometrial stromal sarcoma\nO Reich and S Regauer\n106\nModern Pathology (2004) 17, 104–108\n\ntissues such as the skin, muscle and fat. 4 Also,\naromatase-positive tumor cells may produce estro-\ngen by converting androgens. Immunohistochemical\ndemonstration of aromatase expression may aid in\nidentifying tumor cells utilizing this pathway of\nestrogen production in low-grade endometrial stro-\nmal sarcomas. The heterogeneous staining pattern\nindicates that only a subpopulation of tumor cells in\nlow-grade endometrial stromal sarcomas is capable\nof estrogen production.\nAnalysis of intratumoral aromatase expression in\nlow-grade endometrial stromal sarcomas may shed\nlight on the development and biological behavior of\nthese tumors and may have implications for clinical\nmangement. Aromatase inhibitors are now widely\nused in treatment of estrogen-dependent breast\ncancer.14,23,24 Aromatase inhibitors reduce estrogen\nlevels by inhibiting estrogen synthesis in both tumor\ntissues and peripheral sites, and as a consequence\nthey inhibit the proliferation of the tumor. Aroma-\ntase inhibitors may also influence apoptosis. 24\nAromatase inhibitors are being studied for other\nhormone-dependent benign and malignant tu-\nmors.24,25 The demonstration of aromatase expres-\nsion in low-grade endometrial stromal sarcomas\nmay be a rationale for a study of aromatase\ninhibitors in the treatment of women with low-\ngrade endometrial stromal sarcomas. Spano et al 26\nhave reported initial results of such adjuvant\nhormonal therapy. In their series, two patients with\nmetastatic low-grade endometrial stromal sarcomas\nhad a complete response after treatment with an\naromatase inhibitor.\nIn conclusion, our results demonstrate that most\nlow-grade endometrial stromal sarcomas express\naromatase. Aromatase expression has been shown\nto be associated with tumor growth, maintenance\nand progression and may also play a role in the high\nrecurrence rate of low-grade endometrial stromal\nsarcomas. Aromatase inhibitors appear to represent\nan intriguing novel treatment modality of low-grade\nendometrial stromal sarcomas to reduce growth\nstimulatory effects of estrogens in this entity.\nAcknowledgements\nWe thank Dr N Harada of The First Department of\nBiochemistry (Fujita Health University Hospital,\nToyaoake, Japan) for kindly providing his anti-\naromatase antibody. The authors are grateful to\nDr F Nogales, University of Granada, Spain for\ncontributing a case and Dr K Tamussino for editing\nthe manuscript.\nReferences\n1 Koss LG, Spiro RH, Brunschwig A. Endometrial\nstromal sarcoma. Surg Gynecol Obstet 1965;121:\n531–537.\n2 Evans HL. Endometrial stromal sarcoma and poorly\ndifferentiated endometrial sarcoma. Cancer 1982;50:\n2170–2182.\n3 Reich O, Regauer S, Urdl W, et al. Expression of\nestrogen and progesterone receptors in low-grade\nendometrial stromal sarcomas. Br J Cancer 2000;82:\n1030–1034.\n4 Sasano H, Harada N. Intratumoral aromatase in human\nbreast, endometrial, and ovarian malignancies. Endocr\nRev 1998;19:593–607.\n5 Miller WR, Mullen P , Telford J, et al. Clinical\nimportance of intratumoral aromatase. Breast Cancer\nRes Treat 1998;49:27–32.\n6 Sumitani H, Shozu M, Segawa T, et al. In situ estrogen\nsynthesized by aromatase P450 in uterine leiomyoma\ncells promotes cell growth probably via an autocrine/\nintracrine mechanism. Endocrinology 2000;141:\n3852–3861.\n7 Zhang Z, Yamashita H, Toyama T, et al. Semi-\nquantitative immunohistochemical analysis of aroma-\ntase expression in ductal carcinoma in situ of the\nbreast. Breast Cancer Res Treat 2002;74:47–53.\n8 Tseng L, Tseng JK, Mann WJ, et al. Endocrine aspects of\nhuman uterine sarcoma: a preliminary study. Am J\nObstet Gynecol 1986;155:95–101.\n9 Benedet JL, Pocorelli S. Staging Classification and\nClinical Practice Guidelines of Gynecologic Cancers.\nElsevier: Oxford, 2000.\n10 Harada N. Novel properties of human placental\naromatase as cytochrome P-450: purification and\ncharacterization of a unique form of aromatase.\nJ Biochem (Tokyo) 1988;103:106–113.\n11 Yang S, Fang Z, Suzuki T, et al. Regulation of\naromatase P450 expression in endometrioitic and\nendometrial stroma cells by CCAAT/enhancer binding\nproteins (C/EBPs): decreased C/EBP b in endometriosis\nis associated with overexpression of aromatase. J Clin\nEndocrinol Metab 2002;87:2336–2345.\n12 Balun SE, Yang S, Fang Z, et al. Role of aromatase in\nendometrial disease. J Steroid Biochem Mol Biol\n2001;79:19–25.\n13 Watanabe K, Sasano H, Harada N, et al. Aromatase in\nhuman endometrial carcinoma and hyperplasia. Im-\nmunohistochemical, in situ hybridisation, and bio-\nchemical studies. Am J Pathol 1995;146:491–500.\n14 Genazzani AR, Gadducci A, Gambaccini M. Contro-\nversial issues in climacteric medicine II: hormone\nreplacement therapy and cancer. Maturitas 2001;40:\n117–130.\n15 Horowitz K, Rutherford T, Schwartz PE. Hormone\nreplacement therapy in women with sarcomas. CME J\nGynecol Oncol 1996;1:23–29.\n16 Press MF , Scully RE. Endometrial ‘sarcomas’ compli-\ncating ovarian thecoma, polycystic ovarian disease and\nestrogen therapy. Gynecol Oncol 1985;21:135–154.\n17 Baggish MS, Woodruff JD. Uterine stromatosis: clin-\nicopathologic features and hormone dependency.\nObstet Gynecol 1972;40:487–498.\n18 Gloor E, Schneyder P , Cikes M, et al. Endolymphatic\nstromal myosis. Surgical and hormonal treatment of\nextensive abdominal recurrence 20 years after hyste-\nrectomy. Cancer 1982;50:1888–1893.\n19 Krieger PD, Gusberg SB. Endolymphatic stromal\nmyosis—a grade I endometrial sarcoma. Gynecol\nOncol 1973;1:299–313.\n20 Chang KL, Crabtree GS, Lim-Tan SK, et al. Primary\nuterine endometrial stromal neoplasms. A clinico-\nAromatase in endometrial stromal sarcoma\nO Reich and S Regauer\n107\nModern Pathology (2004) 17, 104–108\n\npathologic study of 117 cases. Am J Surg Pathol\n1990;14:415–438.\n21 Clement BC, Young RH. Tumors and Tumor-like\nLesions of the Uterine Corpus and Cervix. Churchill\nLivingstone: New York, 1993.\n22 Styron SL, Burke TW, Linville WK. Low-grade\nendometrial stromal sarcoma recurring over three\ndecades. Gynecol Oncol 1989;35:275–278.\n23 Brueggemeier RW. Aromatase, aromatase inhibitors,\nand breast cancer. Am J Ther 2001;8:333–344.\n24 Sasano H, Sato S, Ito K, et al. Effects of aromatase\ninhibitors on the pathology of human breast, endome-\ntrial and ovarian carcinoma. Endocr Relat Cancer\n1999;6:197–204.\n25 Noble LS, Simpson ER, Johns A, et al. Aromatase\nexpression in endometriosis. J Clin Endocrinol Metab\n1996;81:174–179.\n26 Spano JP , Soria JC, Kambouchner M, et al. Long term\nsurvival of patients given hormonal therapy for metastatic\nendometrial stromal sarcoma. Med Oncol 2003;20:87–93.\nAromatase in endometrial stromal sarcoma\nO Reich and S Regauer\n108\nModern Pathology (2004) 17, 104–108","source_license":"CC0","license_restricted":false}