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Aromatase in Human Endometrial Carcinoma and Hyperplasia. Immunohistochemical, in Situ Hybridization, and. Biochemical Studies. Keiko Watanabe ...
American Journal ofPathology, Vol. 146, No. 2, February 1995 Copyright © American Society for Investigative Pathology

Aromatase in Human Endometrial Carcinoma and Hyperplasia Immunohistochemical, in Situ Hybridization, and Biochemical Studies

Keiko Watanabe,* Hironobu Sasano,t Nobuhiro Harada,* Masahiko Ozaki,§ Hitoshi Niikura,* Shinji Sato,* and Akira Yajima* From the Departments of Obstetrics and Gynecology* and Pathology,t Tohoku University of Medicine, Sendai, Japan; the Department of Molecular Genetics,* Institute for Comprehensive Medical Science, School of Medicine, Fujita-Gakuen Health University, Toyoake, Japan; and Nippon Kayaku Co.,5 Tokyo, Japan

The expression ofP450 aromatase and other steroidogenic enzymes were evaluated in 42 endometrioid endometrial carcinomas, 23 endometrial hyperplasias, and 7 normal endometrial specimens. These findings were correlated with clinicopathologicalfindings to elucidate the possible biological significance of in situ estrogen production in the development of human endometrial carcinoma. Only weak aromatase immunoreactivity was observed in vascular walls and myometrial ceUs. In contrast, strong aromatase stromal immunoreactivity was observed in 28 of 42 (66.7%) endometrial carcinomas. However, no stromal immunoreactivity was seen in normal or hyperplastic endometrial specimens. Immunoreactivity in the carcinoma stromal ceUs was signfifcantly increased at sites of invasion. These aromatase-positive ceUs were immunohistochemicaly negative for other steroidogenic enzymes involved in estrogen biosynthesis. In situ hybridization studies revealed aromatase mRNA hybridization signals in stromal ceUs but not in carcinoma ceUs. The distribution of aromatase mRNA correlated well with the immunohistochemical localization of aromatase enzyme. Quantitation of aromatase activity demonstrated 8.75 ± 2.75 pmol/hour/mg of protein for endometrial carcinomas (22 specimens) and 0.98 ±

1.95 pmol/hour/mg ofprotein for normal endometrial specimens (4 specimens). Aromatase activity was found in both estrogen receptorpositive and -negative endometrial carcinomas. Aromatase did not vary with respect to the menopausal status of patients with endometrial carcinoma. These results suggest that estrogen is produced in situ in endometrial carcinoma but not in benign endometrial lesions. Such localy synthesized estrogen may act on carcinoma ceUs in a paracrinefashion to promote tumor growth. Additional investigations are necessary, but increased aromatase expression in the stromal ceUls of endometrial carcinoma may therefore play an important role in the development of human endometrioid endometrial carcinoma. (Am J Pathol 1995, 146:491-500)

Generally, estrogen has been considered to be one of the promoting factors of endometrial carcinoma, especially of the endometrioid type.1 2 Although the ovary in premenopausal women is the main contributor to circulating estradiol, estrogen biosynthesis after menopause is mainly peripheral, through conversion of androstenedione or C19 steroids from the adrenal glands and ovaries.3 This peripheral process takes place in skin,4 muscle,5 and fat,6 and this conversion is catalyzed by the enzyme complex cytochrome P450 aromatase. Increased peripheral conversion of androgens to estrogens by P450 aromatase can result in elevated serum levels of estrogens. Although estrogen has been invoked as a promoting factor in endometrial carcinoma, there has been no consistent evidence of increased serum estrogen concentrations in women with endometrial cancer.7-9 Accepted for publication November 7, 1994. Address reprint requests to Dr. Keiko Watanabe, Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1 Seiryou-machi, Sendai, Japan 980.

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Tseng et al10 have examined testosterone aromatization in the human endometrium and have detected estrogenic products in both normal and neoplastic endometrial tissues. In addition, Yamaki et al1l1 have demonstrated that aromatase activity is significantly higher in neoplastic endometria than in normal tissues. They concluded that the human endometrium can directly convert androgens to estrogens and that increased aromatization activity can result in increased estrogen concentrations in neoplastic endometria.1 1 Although these in vitro studies have provided important information on estrogen metabolism in human endometrial disorders, it is not known what types of endometrial cells express aromatase activity. The recent development of antibodies and DNA probes against aromatase have made it possible to localize aromatase by immunohistochemistry and in situ hybridization in normal and abnormal human ovary,12 breast,13 and testis,14 demonstrating possible sites of estrogen production in these tissues. We report here on aromatase immunolocalization, in situ hybridization, and enzyme activity in 72 normal and abnormal human endometrial tissues, including 42 endometrioid endometrial carcinomas. These data are correlated with clinicopathological findings and estrogen receptors (ERs) and progesterone receptors (PRs) to elucidate the possible significance of endometrial estrogen production in the development of human endometrial carcinoma. In addition, immu-

nolocalization of other enzymes involved in estrogen biosynthesis was carried out in carcinomas to determine whether other estrogen biosynthetic pathways operate in human endometrial disorders.

Materials and Methods

Specimens Altogether, 42 endometrioid endometrial carcinoma specimens, 19 non-atypical hyperplastic endometrial specimens, 4 atypical hyperplastic endometrial specimens, and 7 normal endometria were collected at Tohoku University Hospital and other affiliated hospitals located in Sendai, Japan. All carcinomas and some hyperplasia specimens were obtained by hysterectomy. Other specimens were acquired from surgical specimens obtained by endometrial curettage. A total of 15 carcinomas were obtained from premenopausal patients (mean age, 43.3 ± 6.3 years; range, 32 to 53 years) and 27 carcinomas were obtained from postmenopausal patients (mean age, 59.4 ± 6.1 years; range, 51 to 75 years). FIGO surgical staging15 revealed 25 stage 1, 9 stage 11, 8 stage l1l, and no stage IV tumors. The histopathological classification was based on the World Health Organization Histological Typing of Uterine Tumors.16 The histopathological characteristics of the tumors and

Table 1. Immunoreactivity and Biochemical Activity of P450 Aromatose and Clinicopathological Parameters of Endometrial Cancer Patients

Immunoreactivity Positive/Total Correlation* P value (%) Menopausal status Premenopause Postmenopause Histology Well differentiated (grade 1) Moderately differentiated (grade 2) Poorly differentiated (grade 3) Clinical stage 11

III Myometrial invasion None