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[CANCER RESEARCH 42, 4812-4814, 0008-5472/82/0042-OOOOS02.00 November 1982] Cytoplasmic and Nuclear Estrogen and Progesterone Receptors in Male Breast Cancer1 Rosemary J. Pegoraro,2 Dharamraj Nirmul, and Septimus M. Joubert Departments of Chemical Pathology ¡P..J. P. , S. M. JJ and Surgery [D. N.¡,University of Natal, P. O. Box 17039, Congella 4013, Durban, South Africa ABSTRACT Cytoplasmic estrogen receptors were detected in 12 of 13 male breast cancer tumors. There was no significant correlation of receptor levels with the age of the patient, size and histológ ica! grading of the tumor, and stage and nodal involvement of the disease. Nuclear estrogen receptors were found in eight of 10 tumors and six of nine patients had tumors positive for Cytoplasmic progesterone receptors, two of which were also found to contain nuclear progesterone receptors. The presence of Cytoplasmic progesterone receptors, in addition to cytoplasmic and nuclear estrogen receptors, may be indicative of truly hormone-dependent tumors in male breast cancer. Treatment offered to patients included surgery, X-ray therapy, chemo therapy, and orchiectomy, but as yet, no general conclusions of the efficacy of the therapeutic regime can be drawn. INTRODUCTION Estrogen receptors have been studied extensively in neo plasms of the female breast, and their measurement is now firmly established as an aid in predicting response to endocrine therapy (19) and as a guide to prognosis (5, 16). In the course of an ongoing study of estrogen and progesterone receptors in female breast cancer, malignant tumor tissue from 13 male patients has been examined. Wherever possible, both CERs3 and NERs have been measured, since it is the nuclear bound receptor that is believed to be necessary for the expression of estrogen-mediated protein synthesis (19). It has been shown in rat uteri that uterine responses and growth following estrogen administration can be correlated to the number of estrogen receptor complexes in the nuclei and to the duration of nuclear binding (1), clearly illustrating the importance of measuring nuclear receptors. Progesterone receptors have been mea sured because their production is thought to be estrogen dependent (13, 17) and their presence is predictive of an improved response to endocrine therapy in females (4, 12). In this paper, the steroid receptor status has been analyzed in relation to the age of the patient, size and histological grading of the tumor, and stage and nodal involvement of the disease, and where available, the response to therapy is presented. MATERIALS AND METHODS Clinical Material. Breast tumor tissue was obtained either by biopsy ' This study was supported by the South African Medical Research Council through the Preclinical Diagnostic Chemistry Research Group. 1 To whom requests for reprints should be addressed. 3 The abbreviations used are: CER, Cytoplasmic estrogen nuclear estrogen receptor; CPR, Cytoplasmic progesterone clear progesterone receptors. Received April 6, 1982: accepted August 10, 1982. 4812 receptor; receptor; NER. NRP, nu or at mastectomy from 13 men. Ten were black, 2 were Asian, and one was white; their ages ranged from 25 to 77 years. In none of the patients was there any clinical evidence of gynecomastia. Two of the patients were classified as Stage II, 7 as Stage III, and 4 as Stage IV according to the American Joint Committee Staging Manual Criteria. Twelve of the tumors were histologically adenocarcinomas; one was a squamous carcinoma. Tumor tissue was placed in 0.15 M NaCI on ice and transported to the laboratory where it was trimmed of fat and assayed immediately or stored in liquid nitrogen. Homogenization of Tissue. Tumor tissue frozen in liquid nitrogen was homogenized using a microdismembrator (Braun, Melsungen, West Germany) as recommended by the European Organisation for Research on Treatment of Cancer Breast Cancer Co-operative Group (7). Chemicals and Reagents. 17/S-[2,4,6,7-3H]Estradiol (85 to 110 Ci/ mmol), [6,7-3H]ORG 2058 (40 to 60 Ci/mmol), and unlabeled ORG 2058 were obtained from the Radiochemical Centre, Amersham, United Kingdom. All other chemicals used were Analar reagent grade. Estrogen Receptor Assay. The assay method used has been de scribed in detail previously (22) and therefore will be outlined only briefly here. The homogenizing buffer used was 20 mw 4-(2-hydroxyethylM-piperazineethanesulfonic acid, 1.5 mw EDTA, and 0.25 mw dithiothreitol, pH 7.4. About 150 mg tumor tissue were pulverized and homogenized in 3 ml buffer and then centrifuged at 700 x g for 10 min to yield a crude cytosol and nuclear pellet. The nuclear pellet was washed twice to remove trapped Cytoplasmic debris. In order to deter mine whether nuclei were free of Cytoplasmic contamination, lactate dehydrogenase activity was measured in 6 different nuclear suspen sions and cytosols. In each case, the lactate dehydrogenase activity detected in the nuclear suspension was negligible or insufficient to account for the level of receptors measured. Portions of the tumor supernatant and nuclear suspension were incubated separately at 4° without shaking for 18 hr with 17/î-[3H]estradiol, serially diluted to give final concentrations in the range 0.1 to 0.8 nw. Additional portions of both fractions were incubated with 0.8 nw 17/H3H]estradiol Plus a 100-fold excess of diethylstilbestrol. Appropriate blanks were included in the cytoplasmic assay. Free and loosely bound ligand was removed by 0.15% dextran-coated charcoal in the cytosols, while nuclear bound radioactivity was separated on Whatman GF/C filters, using a Millipore filtration unit. The data were plotted according to Scatchard (25) and a straight line was calculated by the method of least squares. Receptors were deemed present if the Kd was less than 7 x 10~'° M. Progesterone Receptor Assay. Progesterone receptors were mea sured in the same tumor homogenate as the estrogen receptors. All buffers used were the same as those used for the assay of estrogen receptors with the inclusion of 10% glycerol (v/v) (23). The assay procedure and conditions were identical to those used for estrogen receptors, except that the final concentrations of [3H]ORG 2058 were in the range 0.1 to 2.4 nw. Additional portions from both fractions were incubated with 2.4 nw [3H]ORG 2058 plus a 100-fold excess of unlabeled ORG 2058. Criteria used to determine positivity were similar to those applied to the assay of estrogen receptors except that the Kd was expected to be in the range of 2 to 10 x 10~'° M (2). Protein and DNA Assays. Cytosol protein concentration was mea sured by the method of Lowry ef al. (18). The DNA content of the nuclear fraction was determined by the method of Burton as modified by Katzenellenbogen and Leake (14). CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 1982 American Association for Cancer Research. VOL. 42 Steroid Receptors in Male Breast Cancer RESULTS Estrogen Receptors. Of the 13 patients whose tumors were assayed for CERs, 12 (92%) were found to be positive with results ranging from 36 to 389 fmol/mg protein (Table 1). Ten of these tumors were examined for NERs, of which 8 were positive. Of the 2 which were negative for NERs, one (Patient 5) was the squamous carcinoma and one was the tumor which was negative for CERs. The 12 patients with CER-positive tumors were all over the age of 48 years, while the only patient whose tumor contained no estrogen receptors in either subcellular compartment was aged 25 years. There was, however, no significant correlation of CER levels with age using the linear regression analysis (r = 0.3013; p > 0.1). Similarly, no corre lation could be found between CER values and the size of the tumor (/• = -0.4445; p > 0.1 ) (Table 2). Using the Kendall rank correlation coefficient T, no correlation of any significance could be observed between the CER levels and the stage of the disease (r = -0.2883; p > 0.1), nodal involvement (T = 0.0165; p > 0.5), or the histológica! grade of the tumor (r = 0.1233; p > 0.5) (Table 2). Progesterone Receptors. Tumors from 9 of the patients in this series were assayed for CPRs, which were found in 6 specimens ranging in value from 196 to 3231 fmol/mg protein (Table 1). Only 2 of these specimens showed any evidence of progesterone binding in the nuclei. Both of these tumors were positive for CERs and NERs, as were the 4 other tumors with CPRs only. Two specimens contained estrogen receptors in both cytoplasm and nuclei, but no progesterone receptors, while one tumor contained no receptors at all. Assay Reproducibility. In order to demonstrate the reproducibility of these assays, 11 tumors were assayed on 2 or 3 occasions. The differences in quantitative results varied from 0.02% to 60% possibly due to tumor heterogeneity, but in no instance did the receptor status change from assay to assay. Clinical Response. Various treatment modalities were used in the management of these patients. Primary treatment con sisted of mastectomy and axillary node dissection, X-ray irra diation to the chest wall, and draining lymph nodes and adju vant chemotherapy. Recurrent or metastatic disease was man aged by a variety of treatment regimens which included orchiectomy, chemotherapy, and X-ray therapy. Only 3 patients with metastatic disease agreed to orchiectomy as part of the treatment regimen. Patient 2 had orchiectomy and combination chemotherapy for osseous métastases and his disease showed stabilization for 3 months before he was lost to follow-up. Patient 6, who developed osseous sec ondaries 8 months after primary treatment with mastectomy and X-ray therapy, was further treated by orchiectomy and single-agent p.o. chemotherapy (cyclophosphamide). He has shown stabilization of his metastatic disease for 14 months. Patient 12, who presented with an ulcerating breast tumor and spinal métastases,was treated by orchiectomy and X-ray irra diation to the spinal secondaries. The ulcerated breast lesion healed following orchiectomy, and there was a 50% reduction in the size of the breast tumor. Details of treatment and response of the remaining patients are not given as they did not receive hormonal therapy and therefore are not relevant to this paper. DISCUSSION In common with observations made throughout the world, the incidence of male breast cancer in South Africa is low. This fact considerably impedes the study of steroid receptors in this disease, and in response to an appeal by Everson et al. (8), results obtained in our laboratory from 13 male patients over a 6-year period have been presented. The high prevalence of CER-positive cancers is in agreement with the literature where 74 of 89 tumors are reported to contain CERs (6, 8-10, 24). Ten of our patients were black, and although numbers pre sented here are small, the indication is that there is no variation in the occurrence of receptors among black and white popuTable 2 CEP concentrations and disease characteristics in volve tee stageIIIIVIIIIIIIIIIIIIIIIIVIIIIVIVIIINodal ment231b1b31b1b01b1blb3lb (cm)7x7Multiple size Patient12345678910111213CERs(fmol/mgprotein)2183892161099413433618236010349239Tu lesions5 x56.5x6Ulcération3.5x31 1.53 .Sx x44x55 x66 X4.51 11.51 1.5 x x 1WHOhisto-logicalgradeIIIIIIIIIIII1IIIIIII11IIIIIIAmericanJointComm Table 1 Estradici and progesterone receptors in patients with male breast cancer CERsPatient1 (yr)59 protein218 M)4.32 fmol/mg M)Not DNA 10"'° pro teinNot DNANot done done done doneNot Not BlackBlackBlackBlackAsianAsianWhiteBlackBlackBlackBlackBlackfmol/mg 4.014.106.200.860.960.270.661.061.880.990.58NERsK„(x Not doneNot Not doneNot 2345678910111213Age 506448565352635025755677RaceBlack 3892161099413433618236010349239K„(x10*'° done1250011521340876102301288116712204.392.051.012.553.592.902.690.89CPRsfmol/mg done624Not done0Not done5166181960032310519Ka(x1.971.173.502.172.450.59NPRsfmol/mg done12480000665600Ka(X4.413.34 NOVEMBER 1982 4813 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 1982 American Association for Cancer Research. R. J. Pegoraro et al. lations. This observation has already been made in females with breast carcinoma (22). In female breast cancer, it is well established that the re sponse rate to hormonal therapy among CER-positive patients is approximately 50 to 60% (2, 4,13). There have been several reports demonstrating an improved prediction rate, as high as 77%, in the response of patients whose tumors contained NERs or CPRs in addition to CERs (2, 4, 15). Friedman ef al. (9) have suggested that, since male breast cancer patients respond to orchiectomy and most have tumors with high CER levels, it would be of greater clinical value to identify those patients who are unlikely to respond to endocrine therapy, so that an alternative form of treatment could be initiated promptly. In Table 1, however, it can be seen that, of the 9 tumors assayed for both estrogen and progesterone receptors, 6 (67%) contained an apparently functional estrogen receptor system; i.e., the CERs appeared to have retained the ability to translocate into the nucleus, resulting in the synthesis of the CPR protein. This figure agrees closely with the 68% objective regression among 41 castrated males reported by Trêves (27). In addition, Neifeld et al. (20) reported an objective 62.5% response rate in their 8 patients and their review of the literature showed a response rate of 50 to 70%. It may be that only tumors with CERs, NERs, and CPRs are truly hormone de pendent, while patients whose tumors contain an impaired receptor mechanism may benefit more from an alternative therapy regimen. The significance of the presence of NRPs is, as yet, uncertain. As only one of our patients (Patient 12) had orchiectomy as the only form of systemic therapy, with partial response, gen eral conclusions regarding the response to hormonal treatment in relation to receptor status cannot be made. It was interesting to note, however, that Patient 6, who had a full receptor complement in his tumor, has shown stabilization of his rib and skull métastasesfor at least 14 months, following orchiectomy and p.o. cyclophosphamide. The single specimen of squamous carcinoma of the breast was included for interest. Whether such a tumor could be hormone dependent is undetermined, but low levels of cytoplasmic estrogen receptor have been reported in skin (11 ) and estrogen receptors have been measured in squamous carci noma of the cervix (26). The lack of correlation of CER level with age did not support the findings of Everson ef al. (8) but was in accordance with those of Friedman et al. (9), as was the absence of any correlation between CER status and the stage of the disease or the histological grade of the tumor. A recent review revealed an objective response rate of 48% to treatment with tamoxifen in 31 patients, while another 5 patients exhibited disease stabilization (21 ). The steroid recep tor status of these patients was not known. Considering the psychological effects of orchiectomy and the reluctance of several patients in this study to undergo castration, we would support Becher ef al. (3) in the use of tamoxifen as initial endocrine therapy in advanced male breast cancer. ACKNOWLEDGMENTS We are indebted to the Department of Pathology. University of Natal, for the histological diagnosis and grading of the tumors. 4814 REFERENCES 1. Anderson, J. N., Peck, E. J., and Clark, J. H. Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology. 96. 160-167, 1975. 2. Barnes, D. M., Skinner, L. 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VOL. 42 Cytoplasmic and Nuclear Estrogen and Progesterone Receptors in Male Breast Cancer Rosemary J. Pegoraro, Dharamraj Nirmul and Septimus M. Joubert Cancer Res 1982;42:4812-4814. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/11/4812 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 1982 American Association for Cancer Research.