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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, National Naval Medical Center, Bethesda, Maryland; the Department of Obstetrics and Gynecology, Walter Reed Army Medical Center, Washington, DC; the Department of Cell and Cancer Biology, National Cancer Institute, National Institutes of Health, Rockville, Maryland; and the Department of Obstetrics and Gynecology, Uniformed Services University of Health Sciences, Bethesda, Maryland.
Address reprint requests to: John Elkas, MD 817 10th Street, Unit 103 Santa Monica, CA 90403 jelkas{at}ucla.edu.
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Abstract |
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Methods: Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-
, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques.
Results: Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P < .003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens.
Conclusion: These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.
Tamoxifen is a nonsteroidal triphenylethylene (monophenylstilbene) antiestrogen that is widely used for adjuvant treatment and chemoprevention in breast cancer. Multiple clinical trials have shown tamoxifen to improve recurrence-free interval and overall survival in both pre- and postmenopausal women with breast cancer.1 These trials have provided the basis for studies in the United States and Europe to test the efficacy of tamoxifen as a prophylactic agent for healthy women at high risk for developing breast cancer. Although tamoxifen has long been considered a safe medication with few serious sequelae, recent reports have cited an increased incidence of endometrial cancer in women using tamoxifen.2 Before tamoxifen is widely used as a chemopreventative agent in otherwise healthy patients, there is a clear need to elucidate the mechanism of action of tamoxifen in the reproductive tract to better understand and predict which group of patients are at high risk for the development of endometrial cancer.
The mechanism of tamoxifen-induced carcinogenesis is thought to be due to its estrogenic actions on the reproductive tract.3,4 The estrogenic activity of tamoxifen is cell type–specific in that it stimulates cell growth and the transcription of a number of estrogen-regulated genes in the uterus but not in the breast.4,5 Thus, it is hypothesized that tamoxifen-induced endometrial carcinogenesis may be due, at least in part, to the regulation of the same genes associated with estrogen action. The aim of this study was to determine whether tamoxifen therapy modulated the protein expression of a number of hormonally responsive genes in the reproductive tract. Specifically, estrogen receptor, progesterone receptor, transforming growth factor-, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin protein expression in benign and neoplastic human endometrium were compared by immunohistochemistry with endometrium obtained from tamoxifen-treated patients.
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Materials and Methods |
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Five secretory phase and three proliferative phase hysterectomy specimens from patients not receiving hormonal therapy were analyzed to serve as normal controls. These specimens were obtained from premenopausal patients who underwent surgery for leiomyomata (n = 4), adenomyosis (n = 2), and benign ovarian cysts (n = 2). Atrophic endometrium (n = 7) was obtained from hysterectomy specimens from postmenopausal patients receiving hormone replacement therapy (HRT) undergoing surgery for pelvic relaxation.
Five-micrometer sections of paraffin-embedded tissues were placed onto sialinized slides. Antibodies against adrenomedullin,6 transforming growth factor- (Biotope, Redmond, WA), lactoferrin (Biodesign, Kennebunk, ME), tumor necrosis factor receptor-II (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and estrogen receptor, progesterone receptor, and Ki-67 (Dako Corp., Carpinteria, CA) were used for immunohistochemistry according to the manufacturers’ specifications. Negative controls included omission of the primary antibody and incubation with nonimmune serum. Background immunostaining was found to be negligible when nonimmune serum was substituted for specific antiserum. Lactoferrin, transforming growth factor-, and tumor necrosis factor receptor-II antibody specificity was confirmed by preabsorption of the antibodies with corresponding purified proteins or peptides (1 µg of peptide to 1:250 dilution of antibody) overnight at 4C followed by centrifugation at 100,000 x g before immunostaining. The preabsorption with purified antigen significantly attenuated tissue immunostaining (data not shown). Immunostaining was visualized by the avidin–biotin–peroxidase complex method using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) with 2,2'-diaminobenzidine as a substrate. Hematoxylin was occasionally used to counter-stain the nuclei of the specimens after immunostaining.
A pathologist blinded to the clinical data analyzed and graded all specimens using standard International Federation of Gynecology and Obstetrics guidelines. The proliferative index and steroid receptor expression were expressed as the percentage of nuclei positive for estrogen receptor/progesterone receptor/Ki-67 expression per total number of cells counted, with at least 500 epithelial cells evaluated for each specimen, at x 400 magnification.
Quantitation of the immunostaining intensity for estrogen receptor, progesterone receptor, adrenomedullin, transforming growth factor-, lactoferrin, and tumor necrosis factor receptor-II was performed by image analysis using the Image-Pro Plus Analysis System (Media Cybernetics, Silver Spring, MD). The system consists of an integrated central processing unit, image monitors, a microscope, and a 3-CCD (U-TV1X) video camera (OPELCO, Sterling, VA). Upon calibration of the system to detect immunoreactive cells, the relative optical density per unit area was determined using the Image-Pro Plus software (Media Cybernetics). For each sample, nine separate areas on the specimen were analyzed and the relative optical density values obtained were evaluated by determining the mean and standard error (SE). Data were analyzed using Kruskal–Wallis analysis of variance, with post hoc pairwise comparisons using the Mann–Whitney test. With a Bonferroni correction for the multiple pairwise tests, P < .003 was considered significant.
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Results |
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) and progesterone receptor (Figure 1B), which correlated with an elevated Ki-67 expression (Figure 1C). The epithelium of secretory endometrium demonstrated downregulation of both the estrogen receptor (Figure 1E) and progesterone receptor (Figure 1F), as well as a decreased proliferative index, although enhanced DNA synthesis was evident in scattered stromal cells (Figure 1G). Atrophic endometrial specimens from patients receiving HRT showed variability in the number of glandular epithelial cells positive for estrogen receptor (Figure 1I) and progesterone receptor (Figure 1J), but all were consistently characterized by a low proliferative index of glandular epithelial cells (Figure 1K). Quantitation of the immunostaining confirmed the observed differences in the level of expression for the estrogen receptor, progesterone receptor, and proliferative index among proliferative, secretory, and atrophic endometrial specimens (Table 1).
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) expression approaching the levels found in proliferative endometrial specimens (Table 1). Progesterone receptor (Figure 1N) expression in tamoxifen-treated patients was also elevated compared with secretory and atrophic controls but did not reach the levels seen in proliferative endometrium. Although the degree of growth exhibited by benign tamoxifen specimens (Figure 1O) was slightly lower than proliferative endometrium, it was significantly elevated compared with secretory and atrophic controls (P < .003).
Analysis of the well-differentiated endometrial adenocarcinomas from tamoxifen-treated patients also demonstrated high levels of estrogen receptor (Figure 1Q) and progesterone receptor (Figure 1R) protein expression. These levels were similar to those seen in proliferative endometrium and benign endometrium from tamoxifen-treated patients (Table 1). As expected, the proliferative index of the malignant tamoxifen-treated endometrium (Figure 1S) was similar to that seen in the proliferative specimens. There were no significant differences in the proliferative index, estrogen receptor, or progesterone receptor gene expression between tamoxifen-treated and untreated malignant epithelium.
Cyclic variation expression of adrenomedullin protein was observed in the endometrium. Proliferative endometrium (Figure 1D) displayed intense cytoplasmic staining, whereas secretory endometrium (Figure 1H) exhibited more heterogeneous immunostaining for adrenomedullin. Atrophic endometrium (Figure 1L) demonstrated variable staining patterns for adrenomedullin often intermediate to that found in the proliferative and secretory phases. Benign endometrium from tamoxifen-treated patients (Figure 1P) demonstrated adrenomedullin expression in the epithelium at levels approaching that observed in proliferative endometrium (Table 1). Regardless of tamoxifen exposure, adenocarcinomas displayed intermediate levels of adrenomedullin protein expression (Figure 1T).
Transforming growth factor- expression in the endometrium of tamoxifen-treated patients was similar to that found during the proliferative phase (Table 1). Overall, proliferative phase endometrium (Figure 2A) and benign tissue from tamoxifen-treated patients (Figure 2J) exhibited higher levels of transforming growth factor- than found in secretory endometrium (Figure 2D), atrophic endometrium (Figure 2G), or the adenocarcinomas regardless of tamoxifen administration (Figure 2M).
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) and HRT (Figure 2H). Malignant tissue from tamoxifen-treated patients (Figure 2N) had significantly higher levels of protein expression compared with malignant tissue from patients not treated with tamoxifen (P < .003). Significant differences in tumor necrosis factor receptor-II expression (P < .001) were also noted between proliferative (Figure 2B) and secretory endometrium, (Figure 2E) confirming previous reports of cyclic variation of tumor necrosis factor receptor-II expression.7
High levels and distinctive patterns of immunostaining for lactoferrin were noted in atrophic endometrium (Figure 2) compared with other specimens. The proliferative (Figure 2C) and secretory (Figure 2F) specimens exhibited weak lactoferrin immunostaining, as did the benign tamoxifen specimens (Table 1). Furthermore, lactoferrin was present in only a small population of glands in these samples and was not always detectable in every patient. Consistent with previously published data, well-differentiated adenocarcinomas, regardless of tamoxifen exposure (Figure 2O), showed high levels of lactoferrin expression compared with proliferative, secretory, and benign tamoxifen-treated endometrium (Table 1).8
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Discussion |
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, lactoferrin, adrenomedullin, and tumor necrosis factor receptor-II are expressed in the endometrium, suggesting that these factors may play a role in the growth and differentiation of the endometrium in response to steroid hormones.7–10 Based on these studies, we hypothesized that modulation of these genes in the endometrium may be associated with the physiologic response to tamoxifen in the uterus and may contribute to the development of hyperplastic and neoplastic lesions.
We have shown that tamoxifen stimulates a number of estrogen-regulated genes in the endometrium, mimicking, in part, the gene expression of proliferative endometrium. Specifically, proliferative and benign tamoxifen-treated endometrium had similar levels of estrogen receptor, progesterone receptor, adrenomedullin, transforming growth factor-, lactoferrin, and tumor necrosis factor receptor-II protein expression. However, tamoxifen’s ability to stimulate epithelial growth (Ki-67) was significantly less than found in proliferative endometrium, although it was markedly elevated over secretory and atrophic endometrium. This may indicate that tamoxifen does not act as a full estrogen agonist in the endometrium. Instead, there may be an uncoupling of the growth response from the induction of growth regulatory or proliferative phase genes and the classical estrogen markers, estrogen receptor and progesterone receptor.
Our observation of tamoxifen’s effects on steroid receptors in the endometrium supports, in part, the findings of Schwartz et al, who also noted high levels of progesterone receptor expression in tamoxifen-treated women compared with atrophic controls.3 Unlike Schwartz et al, however, we did find a significant increase in estrogen receptor levels in tamoxifen-exposed tissue compared with atrophic endometrium. This difference may be because our atrophic samples were taken from women receiving HRT, whereas the patients were not hormonally treated.
The most significant difference between the tumors and the benign specimens was the proliferative index. The tumors, regardless of tamoxifen exposure, had proliferative indices comparable to proliferative endometrium. In addition, compared with endometrium from premenopausal and tamoxifen-treated patients, the tumors exhibited a greater number of lactoferrin positive cells, consistent with previous observations.8 Unexpectedly, the atrophic endometrium of postmenopausal patients receiving HRT contained significantly greater levels of lactoferrin than found in any other specimen, which makes the usefulness of lactoferrin as a marker for malignant disease limited in postmenopausal patients.
Our comparative analysis of the endometrial adenocarcinomas failed to demonstrate significant differences between tamoxifen-treated and untreated samples other than elevated tumor necrosis factor receptor-II levels in the tamoxifen specimens. This significance of this is unclear. Other studies have shown that tumor necrosis factor receptor-II gene expression is markedly elevated in endometrial adenocarcinomas, especially those of high grade.7 In addition, animal studies have shown that estrogen may be the primary regulator of tumor necrosis factor receptor-II in the uterus.11 An association of tumor necrosis factor receptor-II with estrogen is also suggested in our study, in that the proliferative endometrium expressed greater levels than the secretory endometrium, and that the endometrium of both HRT- and tamoxifen-treated patients expressed levels of tumor necrosis factor receptor-II similar to proliferative endometrium. This suggests that tamoxifen behaves as an agonist for the regulation of tumor necrosis factor receptor-II and the threshold for induction of tumor necrosis factor receptor-II is low, because the low estrogen levels associated with either HRT or the mixed agonist/antagonist activity of tamoxifen is sufficient. Further study with a larger sample size is necessary to determine whether tamoxifen selectively upregulates the tumor necrosis factor receptor-II system in the endometrium.
Our data provide support for the hypothesis that the stimulation of genes characteristic of proliferative endometrium may play a role in tamoxifen-induced endometrial pathology. However, we have shown that the induction of these genes does not necessarily translate into enhanced growth potential. There appears to be an uncoupling between tamoxifen’s ability to induce genes associated with proliferative endometrium and its ability to induce growth. The diversity of response of the endometrium to tamoxifen may be related to differential activation of estrogen-target genes, and for most patients tamoxifen will behave as only a partial agonist with limited ability to regulate genes that directly control proliferation.
Of the plethora of prognostic markers that may be useful for evaluating the estrogenic effects of tamoxifen in the endometrium, the analysis of the estrogen receptor, progesterone receptor, and proliferative index may indicate whether tamoxifen is behaving as either a partial or full agonist, and thus aid in identifying patients at greatest risk for developing carcinoma.
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Footnotes |
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Funding for the study was provided by the Department of Clinical Investigation, Walter Reed Army Medical Center and the Research Administration, Uniformed Services University of the Health Sciences.
Received May 10, 1999. Received in revised form October 19, 1999. Accepted October 27, 1999.
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References |
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2. Fisher B, Costantino JP, Redmond CK, Fisher ER, Wickerham DL, Cronin WM. Endometrial cancer in tamoxifen-treated breast cancer patients: Findings from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J Natl Cancer Inst 1994;6:527–37.
3. Schwartz LB, Krey L, Demopoulos R, Goldstein SR, Nachtigall LE, Mittal D. Alterations in steroid hormone receptors in the tamoxifen-treated endometrium. Am J Obstet Gynecol 1997;176:129–37.[Medline]
4. Webb P, Lopez GN, Uht RM, Kushner PJ. Tamoxifen activation of the estrogen receptor/AP-1 pathway: Potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 1995;9:443–56.[Abstract]
5. Ramkumar T, Adler S. Differential positive and negative transcriptional regulation by tamoxifen. Endocrinology 1995;136:536–42.[Abstract]
6. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al. Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 1996;137:2626–32.[Abstract]
7. Terranova PF, Hunter VJ, Roby KF, Junt JS. Tumor necrosis factor-alpha in the female reproductive tract. Proc Soc Exp Biol Med 1995;209:325–42.[Abstract]
8. Walmer DK, Padin CJ, Wrona MA, Healy BE, Bentley RC, Tsao MS, et al. Malignant transformation of the human endometrium is associated with overexpression of lactoferrin messenger RNA and protein. Cancer Res 1995;55:1168–75.
9. Gray K, Bullock B, Dickson R, Raszmann K, Walmer D, McLachlan J, et al. Potentiation of diethylstilbestrol-induced alterations in the female reproductive tract by transforming growth factor-transgene expression. Mol Carcinog 1996;17:163–73.[Medline]
10. Montuenga LM, Martinez A, Miller MJ, Unsworth EJ, Cuttita F. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 1997;138:440–5.
11. Roby K, Hunt J. Mouse endometrial tumor necrosis factor-alpha mRNA and protein: Localization and regulation by estradiol and progesterone. Endocrinology 1994;135:2780–9.[Abstract]
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