Journal of Surgical Oncology 7:467-477 (1 975)
Estrogen and Estrogen Receptors of Breast .......................................................................................... .......................................................................................... VELAYUDAN SAHADEVAN, M.D., Ph.D., CHARLES P. PERLIA, M.D., STEVEN G. ECONOMOU, M.D., and HOWARD SKY-PECK, Ph.D. Human breast cancer can be divided into a group that contains specific receptor sites for estrogen and a group without such specific estrogen-binding sites. The presznce of specific estrogen receptors in some tumors indicating hormonal dependency has been shown t o be of predictive value for endocrine treatment. This would greatly improve therapeutic planning for patients with breast cancer. Tumor tissue from 52 patients was investigated for content of both cytosol estrogen and estrogen receptor. I n addition, the total tumor estrogen was also determined in 14 of these tumors. The results of this investigation show two distinct groups: one group containing both estrogen receptor and estrogen and a second group with n o receptor but with measurable amount of estrogen. Tumors with estrogen receptors have higher tissue levels of estrogen than tumors without specific estrogen receptor. Even in the absence of estrogen receptor, however, most tumor tissue examined contained a measurable amount of estrogen.
........................................................................................... ......................................................................................... KEY WORDS: breast cancer, estrogen, estrogen receptor, estrogen antiserum, total tumor estrogen, cytosol estrogen
INTRODUCTION When tritium labeled estradiol was injected into patients with metastatic breast cancer prior to adrenalectomy, the cancer of patients responding to adrenalectomy was found to concentrate radioactive estrogen more intensively than the metastatic tissue of nonresponding patients (Folca et al., 1961). This was followed by the observation that dimethylbenzanthracene (DMBA)-induced hormone-dependent rat mammary tumors had higher in vitro uptake of radioactive estrogen that did autonomous tumors (Mobbs, 1966). It was further demonstrated that the preferential uptake of estrogen by hormone-dependent breast tumors could be inhibited by synthetic analogous of estrogen (Jensen et al., 1967). Subsequently, it was shown that the uptake of estrogen by cancer tissue depended on the presence of a specific estrogen-binding protein in the cytoplasm of such tissue (Jensen et al., 1967; Mobbs 1966). This high affinity of estrogen-binding protein is known as Section of Medical Oncology, Department of Medicine, Department of General Surgery and Department of Biological Chemistry, Presbyterian-St. Luke’s Hospital, Chicago, Iliinois. Address all correspondence t o Dr. V. Sahadevan, Presbyterian-St. Luke’s Hospital, 175 3 West Congress Parkway, Chicago, Illinois 60612.
467 0 197.5 A l a n R . Liss, I n c . , 1.50 Fifth A v e n u e , New York, N . Y . 10011
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estrogen receptor. The physical characteristics of this estrophilic protein are shown to be an 8s and/or 4s protein (Jensen and DeSombre, 1973; McCuire and Chamness (1973). When breast tumor tissue contains estrogen-receptor protein, the tumor nucleus is also known to accumulate estrogen (McGuire and Julian, 1971). Nuclei from tumors that are devoid of estrophilic cytoplasmic receptors do not have the ability to concentrate estrogen. On the other hand, if such tumors are incubated with estrogen receptors of uterus, the tumor nuclear binding of estrogen can be readily established (McGuire et al., 1972). It is obvious therefore, that the nucleus of tumor tissue that does not contain estrogen receptor can be induced t o bind estrogen in the presence of exogenous estrogen-receptor protein. Human breast tumors show a wide range of estrogen-receptor proteins (McGuire 1973; Korenman and Dukes, 1970; Feherty et al., 1971; Wittliff et al., 1972; DeSombre et al., 1974). It is reported that up to about 50% of breast tumors have measurable amounts of estrogen-receptor protein (DeSombre et al., 1974). The frequency of estrogenreceptor proteins in tumors of postmenopausal patients is considerably higher than in tumors of premenopausal patients (DeSombre et al., 1974). Usually the receptor concentration of the tumor does not decrease during the progression of the disease. Occasionally, however, the metastasis of the same patient may be with and without estrogen receptor (DeSombre et al., 1974). Tumors that are shown to have estrogen-receptor sites are known to respond to such endocrine treatments as oophorectomy, adreanalectomy, or additive endocrine therapy (Jensen et al., 1971; McGuire et al., 1974). The rate of response to endocrine treatment of patients with tumors that contained estrogen receptors was about 77%. The response rate was about 3% in patients with tumors devoid of estrogen receptor. (Jensen et al., 1971). Similar reports on tumor regression and its relationship to estrogen receptor have been reported by several other independent study groups (Savlov et al., 1974; Mass et al., 1972; Braunsberg et al., 1973). It has been suggested that the variation of receptor concentrations in tumors may be related to tumor contents of epithelial and nonepithelial cells (McCuire et al., 1974). No correlation, however, is found between the histology of a tumor and its estrogen receptor concentration (Wittliff et al., 1972). An alternative hypothesis is that in the presence of endogenous tumor estradiol only a few unoccupied receptor sites would be available for the binding of added exogenous radioactive estrogen (McGuire et al., 1974). It is important, therefore, t o determine the tissue levels of estrogen of tumors with and without estrogen receptor. Saturation of all estrogen receptors with endogenous estrogen may give false negative results. The investigations reported in this est rogen-receptor assay paper were undertaken to assess the relationship of estrogen receptor and tissue levels of estrogen of tumors with and without estrogen receptors. MATERIALS AND METHODS Patient Selection Surgical specimens were obtained from patients referred to the Presbyterian-St. Luke’s Hospital for the management of primary or metastatic breast cancer. Both estrogenreceptor and tumor-estrogen determinations were made on tissue obtained at the time of corrective mastectomy or when it was obtained to confirm recurrence of disease. The patients were evaluated and followed in close cooperation between the surgeon and medical oncologist.
Estrogen and Estrogen Receptors of Breast Cancer
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Radioimmunoassay of Tumor Tissue Contents of Estrogen Antiserum to estradiol-17 beta. Conjugate of bovine serum albumin with estradiol17 beta was prepared by carbodiimide reaction (Gross, 1970). This congugate, together with the complete Freund’s adjuvant and physiologic saline, was used for the immunization of sheep. After several subcutaneous injections of this antigen prepared at weekly intervals, blood was drawn and the antiserum tested for its ability t o bind with radioactive estradiol 17 beta. At an effective dilution of 1:200,000 this antiserum binds about 50% of the added radioactive estradiol-17 beta. Crossreaction of estradiol-17 beta antiserum with other related steroids. The antiserum against estradiol-17 beta cross-reacts with estrone almost with the same affinity as the estradiol-17 beta. At an effective antiserum dilution of 1:200,000, almost identical binding of both estradiol-17 beta and estrone are observed. The magnitude of crossreaction of this antiserum with the estriol was significantly less than with estrone. Crossreaction with the steroid is observed only when the antiserum is diluted to 1:40,000. At this dilution, the antibody affinity to estrone is nearly the same as for estradiol-17 beta. As shown in Fig. 2 , at a higher antiserum dilution of 1 :200,000 its cross-reaction is much less. No cross-reaction of this antiserum is observed with other steroids (Fig. 1). Since no attempts were made to separate the various components of estrogen from tumor tissue extract, the values obtained for tumor estrogen in this study represent the total tissue levels of estrogen. The related antibody affinity to various steroids is shown in Figs. 1 and 2. Extraction of estrogen. The tumor cytosol was prepared as described in the section on estrogen-receptor assay. The cytosol contents of estrogen was extracted with 3 volumes of ethanol. The whole ethanol extraction was evaporated to dryness under a stream of air. The residue was redissolved in phosphate saline buffer prior to radioimmunoassay. The
5
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2m
5m
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Fig. 1. Standard curve showing the inhibition of 3H-estradiol-17 beta binding to antibody by increments of unlabeled estradiol-17 beta. As with testosterone, all other steroids (cholesterol, progesterone, 1 1-alpha-OH-progesterone, pregnenolone, 19-OH-androstendione, dehydroapepiondrosterone, hydrocortisone, and dehydropregnenolone) failed to bind to this antiserum.
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1M
zoo
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ICUI
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Fig. 2. Standard curves showing binding affinity to antibody by various estrogens.
total tissue estrogen was extracted by digesting 100 mg tumor tissue with 0.2 ml 25% HC1 and, after a short period of homogenization, extracting the estrogen twice with 2 ml of ether. The ether extract was evaporated and, prior to radioimmunoassay of estrogen, was redissolved in 0.1 M phosphate saline buffer, pH 7.4. The average estrogen extraction efficiency by these procedures was about 60-70%. Radioimmunoassay procedures Standard curve. Into duplicate siliconized glass tubes, estradiol- 17 beta dissolved in ethanol was added at a range of 2.5 to 500 pg and the ethanol evaporated under a stream of air. Into such tubes there was added 0.1 ml of 1:100,000 antiserum dilution and about 10 pg 3H-estradiol-17 beta in 0.1 ml volume. The dilutions of antiserum and radioactive estradiol were made in phosphate buffer containing 0.1%gelatine as a carrier protein. The tubes were incubated for 12 hr at 4°C. The separation of antibody bound and unbound 3H-estradiol-17beta was done by the addition of 0.1 ml of a 1.5% dextran-coated charcoal in phosphate buffer (Hotchkiss et al., 1971). The tubes were then centrifuged and the supernatant containing the antiserum bound radioactive estrogen measured by liquid scintillation counting. The lowest measurable amount of estradiol- 17 beta by this procedure was found to be 2.5. Estimation of estrogen content of specimens. As in the preparation of the standard curves, to the tubes containing estrogen extracted from the tissue, both antiserum and 3Hestradiol-I7 beta were added after evaporation of its ether contents. After incubation for 12 hr, the bound and unbound radioactive estrogen were separated and the radioactivity measured as described above. Since the percentage of antibody bound estrogen is proportional to the nonradioactive estrogen content of the specimen, the tissue levels of estrogen were calculated from the standard curve and corrections made for the extraction efficiency. The efficiency of the assay system. The recovery of added nonradioactive estrogen to tissue, its extraction, and its subsequent radioimmunoassay were used as parameters to test the efficiency of this assay. As shown in Fig. 3, the recovery, when adjusted to the
Estrogen a n d Estrogen Receptors of Breast Cancer
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Fig. 3. Recovery of nonradioactive estrogen added to tumor tissue. Increments of nonradioactive estrogen were added to tumor and the estrogen extracted and assayed. When adjusted to extraction efficiency, the recovery experiments run parallel with the standard curves constructed with the same amount of added nonradioactive estrogen.
extraction efficiency, runs parallel with the standard curve constructed with the same amounts of added nonradioactive estrogen. Estrogen receptor assay. Immediately after removal of the cancerous tissue, the specimen was taken to the laboratory and kept on crushed ice. After removing fat, necrotic tumor, and connective tissue, the tumor specimen was either immediately processed for estrogen receptor and estrogen determination or stored frozen. In preparation for tumor cytosol, approximately 0.5-1 gm tumor tissue was pulverized with an automatic frozen tissue pulverizer and subsequently homogenized with the homogenizer PT ST (Brinkman Instruments). Four ml tris-EDTA buffer (0.01 M tris-HC1,0.0015 M EDTA, pH 7.4) was used for 1 gm of tumor tissue for the assay done by the dextran-coated charcoal procedure. Much lesser volume of buffer is used for the assay using sucrose gradient procedures. The homogenate was next centrifuged at 20,000 rpm for 30 min and this supernatant used for the initial dextran-coated charcoal assay. For the sucrose gradient assay procedure, the supernatant was further centrifuged at 150,000 g for 45 min in a Beckman Ultracentrifuge L5-65. During this processing the tissue was kept at about 0°C. Preliminary assay of estrogen receptor by dextran-coated charcoal procedure. The principle of this assay procedure consists of estimation of tumor cytosol bound radioactive estradiol in the presence and absence of either nonradioactive estrogen or its analogue U11, 100 (Nafoxidine) and subsequent separation of bound and unbound Hestradiol by dextran-coated charcoal (Korenman and Dukes, 1970; Leung et al., 1973).
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Because of the molecular similarity of Nafoxidine to estrogen, this compound competes with estrogen for the binding site of receptor. The competitive inhibition of 3H-estradiol binding to receptor protein by Nafoxidine will decrease the estrogen receptor bound radioactivity if the specimen contains specific estrogen receptor. The percentage inhbition of radioactive estrogen binding by Nafoxidine is calculated from the difference in the receptor bound radioactivity in the presence and absence of Nafoxidine. To duplicate tubes, 0.5 ml aliquots of cytosol were added to a mixture of 50 pl of Tris-EDTA buffer with or without 10 p1 of lop4 M Nafoxidine. After 15 min incubation at 0°C 10 pg of 3H-estradiol were added and the mixture incubated at 0°C for an additional 30 min. After incubation the bound and unbound radioactive estrogen was separated with 0.1 ml of a suspension containing 1.5 gm Norite charcoal and 150 mg of Dextran 70/100 ml Tris-EDTA buffer. After adding the charcoal the mixture was incubated for 10 min, the unbound estradiol was adsorbed by the charcoal and then separated by centrifugation. The radioactivity of the bound H-estradiol containing supernatant was measured by liquid scintillation countings in a Packard Automatic Tri-Carb Liquid Scintillation Spectrometer System, model 3330. The specific binding of estrogen by its receptor protein was calculated from the difference in the receptor bound radioactivity in the presence and absence of Nafoxidine. The receptor concentration is expressed as femtomoles (lo-'' moles) 3H-estradiol bound/gm wet weight of tumor. Estrogen receptor assay by sucrose-gradient analysis. Except for the following changes this assay procedure was essentially the same as described by McGuire (1973). About 150 p1 of tumor cytosol prepared as described above were added to a mixture of 50 pl of Tris-EDTA buffer with or without 10 p1 of lop4 M Nafoxidine. After 15 min of incubation at O"C, 10 pg of 3H-estradiol were added to the mixture and incubated at 0°C for an additional 30 min. After incubation, the unbound 3H-estradiol was removed with 0.1 ml of a 1.5% solution of dextran-coated charcoal and subsequent centrifugation to remove the charcoal. Approximately 200 pl of the supernatant were layered onto a cold linear 5-20% sucrose gradient that was prepared in the same buffer as the cytosol. The gradient was then centrifuged at 44,000 rpm for 15 hr in a Beckman ultracentrifuge L5-65. After centrifugation, 0.3 ml fractions were collected by puncturing the bottom of the tube, and the distribution of radioactivity was measured by liquid scintillation countings. The specific estrogen-receptor binding of H-estradiol was identified by isotopic profiles from sucrose gradient and differences in the bound radioactivity at the 8s or 4s region in the presence and absence of Nafoxidine. Figure 4 illustrates the specific estrogen-receptor binding of H-estradiol and its inhibition by both Nafoxidine and nonradioactive estradiol. RESULTS Tumor Estrogen All 52 tumor specimens were examined for their cytosol estrogen concentration. Sufficient tumor tissue from only 14 patients was available for the estimation of total tissue contents of estrogen. Tumors of postmenopausal patients in general had lower tumor estrogen, particularly if they did not have estrogen receptor. Among the 20 specimens without estrogen receptor (from postmenopausal patients), 9 had no measurable cytosol estrogen. The average cytosol estrogen level of these patients was 82 pg per gm wet weight of tumor tissue. On
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Fig. 4. Sucrose gradient assay of estrogen receptors. Both Nafoxidine (NAF) and nonradioactive estrogen (E2) inhibits the 3H-estradiol binding. Since this competitive inhibition is observed both at 8s region (bottom of the tube) and at 4s (top of the tube) this tumor specimen contains both 8s and 4s receptor protein.
the other hand, of the 19 with estrogen receptor from postmenopausal patients, only 5 of the tumor cytosols were devoid of estrogen. The remaining 14 had a relatively higher cytosol estrogen level; the average was 135 pg per gm wet weight of tumor. The tumor cytosol contents of estrogen in premenopausal patients was in general much higher even if these tumors were negative for estrogen receptor. The tumors of 10 premenopausal patients that were negative for estrogen receptor had an average of 145 pg estrogen per gm tumor tissue. This is nearly twice the cytosol estrogen contents of similar patients with tumors negative for estrogen receptor. In pre- and postmenopausal patients the cytosol estrogen content of tumors with estrogen receptor did not show any significant difference; their average values were 133 and 135 pg per gm wet weight of tumor, respectively. These results are summarized in Tables I and 11. The cytosol content of estrogen, however, may not be a true reflection of total tumor contents of estrogen. A greater part of the estrogen may be bound to the nucleus (McGuire and Julian, 1971). On 14 tumors, both cytosol and total estrogen contents were estimated and their reiationship to estrogen receptor assessed (Table 111). The estrogen contents of tumors of preand postmenopausal patients were significantly different in the presence of estrogen
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Endogenous Tumor Cytosol Estrogen of Postmenopausal Patients
TABLE I.
Receptor Positive No. 1 2 3 4 5 6
I 8 9 10 11 12 13 14 15 16 17 18 19
Patient Ro Kr Hu cog
La Ma HaC Col He Sa Qu Ke HaH Ta Jo ThE Ku BeJ WoA
Average
Receptor f moles/gm
Receptor Negative Estrogen pg/g
No.
Patient We Kil Kiz SchM
88 71 4 24 57 174 I06 315 45 178 112 232 22 22 58 19 40 59 372 656
96 245 0 0 137 490 158 34 0 25 123 62 78 0 0 57 196 508 323
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
164
135
Average
AnP
SchR Wi Fo Oa Ar
WOE Go ThI: Ad AnE BeV Wh Pe Mu
Estrogen pg/gm
0 0 7 0 10 11 0 0 4 1 0 10 0 0 0 0 0 0 12
239 0 0 20 0 193 435 0 0 0 372 45 100 0 17 59 23 0 0
3
82
Endogenous Tumor Cytosol Estrogen of Premenopausal Patients
TABLE 11.
Receptor Positive Receptor
No.
Patient
f moles/gm
1 2 3
Jo Dr LuD
318 88 270
225
Average
Receptor f moles/gm
Receptor Negative Estrogen pg/gm
Receptor
Estrogen pg/gm
No.
Patient
f moles/gm
52 347 0
1 2 3 4 5 6 7 8 9 10
Ge McB LuN Ba La Pi Sk SchN Br zy
0 0 0 0 0 3 0 0 0 17
209 56 51 173 50 476 103 0 0 335
133
Average
2
145
receptor than when this specific receptor protein was absent. Tumors with estrogen receptors had higher estrogen levels both in tumor cytosol and in total tumor tissue. In the absence of estrogen receptor, however, there was significant estrogen in tumor tissue, and this was much higher in specimens which were taken from premenopausal patients and which did not contain estrogen receptor.
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Estrogen and Estrogen Receptors of Breast Cancer
TABLE 111.
Endogenous Tumor Estrogen and Estrogen Receptor
Estrogen Receptor Positive
No.
1 2 3 4
Patient
Estrogen Receptor Negative
Estrogen Pgk
Receptor f moles/gm of tissue
Cytosol
Total
No.
232 40 656 88
123 57 323 347
652 99 1267 371
1 2 3 4 5 6 7 8 9 10
254
213
597
Qu ThE WoA Dr
Average
Estrogen pg/gm
Patient
Receptor f moles/gm of tissue
Cytosol
Total
Ki SchM Ad AnE BeV Wh No Ge* Sk SchN*
7 0 0 0 0 0 9 0 0 0
0 20 0 17 59 23 148 209 103 0
12 169 263 0 89 175 771 232 303 284
2
58
230
Average
*Premenopausal
Estrogen Receptor Both estrogen receptor and the estrogen content of tumor were investigated in 52 patients. Estrogen-receptor values over 20 f moles per gm wet weight of tumor tissue are arbitrarily taken as positive for estrogen receptor and values below this are considered as negative for estrogen receptor. This value was taken to account for the technical errors involved in the present system of estimations of estrogen receptors. Tumor tissue was positive for estrogen receptor in 22 of the 52 patients. When these patients were grouped according to their menstrual status, there was a distinct difference in the occurrence of estrogen-receptor activity in the tumors of postmenopausal patients. Among the 13 premenopausal patients, in only 3 did the tumor show estrogen receptor. Their estrogen-receptor contents varied from 88 to 318 f moles per gin tumor tissue. On the other hand, among the 39 tumor tissues obtained from the postmenopausal patients, 19 had tumors with estrogen receptor ranging from 22 to 656 f moles. These results are summarized in Tables I and 11.
DISCUSSION As already mentioned, there is an increased recognition of the significance of estrogen receptors in breast tumors. Knowledge of these receptors would greatly improve the ability to plan therapy for patients with breast cancer. The significance, however, of a wide range of receptor concentration in tumors with estrogen receptor is not known. It has been suggested that the endogenous tumor estradiol contents may significantly alter the tumor receptor concentration (McGuire et al., 1974). If all the estrogen-receptor sites of the tumor tissue are occupied by endogenous estrogen, no additional binding of the added radioactive estrogen to the tumor cytosols could be observed. With our present methods of estrogen-receptor assay, only the binding of added radioactive estrogen to tumor cytosol is measured and, therefore, the saturation of receptor protein with
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endogenous hormones may be highly significant in the evaluation of tumor containing estrogen receptor. In a study of 6 patients, it was shown that the endogenous tumor estrogen may not saturate all the binding sites of estrogen receptor (Korenman and Dukes, 1970). In the study reported here, a more sensitive radioimmunoassay for determination of tumor estrogen is employed. The relationship of endogenous tumor estrogen concentration to tumor estrogen receptor is assessed in tumor tissue of 5 2 patients. This study demonstrates that the contents of estrogen in tumor tissue is related to both endogenous estrogen and the tumor content of estrogen receptor. In postmenopausal patients, the tumor estrogen content is markedly enhanced in the presence of estrogen receptor. On the other hand, in tumors of premonopausal women, the presence of estrogen receptor seems not to have an influence on the tumor content of estrogen. This may indicate that the estrogen content of tumor is mostly influenced by the endogenous estrogen. More studies on premenopausal tumors, however, are required for further analysis of this observation. On the other hand, in postmenopausal patients the presence of estrogen receptor is closely associated with a higher endogenous tumor estrogen concentration. This may indicate that in the presence of estrogen receptor, the estrogen utilization by the tumor tissue is enhanced. In conclusion, this study clearly demonstrates that in those tumors with estrogen receptor the wide range of estrogen receptor is unrelated to the endogenous tumor content of estrogen. Contrary to previous speculation, with a higher estrogen receptor in a tumor there is proportionately higher estrogen content. This may be related to the rapid turnover of estrogen in tumors with estrogen receptor. The estrogen binding and the transformation of the estrogen receptor from its 8s to 5s receptor may be very rapid in tumors with estrogen receptor.
ACKNOWLEDGMENTS This work was supported by the Breast Cancer Research Foundation and Public Health Service Grant #4729 1. The authors greatly acknowedge the excellent technica1 assistance of Mrs. Jean Harrison, Mrs. Victoria Sahadevan, and Miss Sirima Silva. REFERENCES Braunsberg, H., James, V. H. T., and Irvine, W. T. (1973). Prognostic significance of’ estrogen uptake by human breast cancer tissue. Lancet 1:163-165. DeSombre, E. R., Smith, S., Block, G. E., Fergusin, D. J., and Jensen, E. V. (1974). Prediction of breast cancer response t o endocrine therapy. Cancer Chemother. Rep. 58:s 13-519. Folca, P. J., Glascock, R. F., and Irvine, W. T. (1961). Studies with Tritium-labeled hexoestrol in advanced breast cancer. Comparison of tissue accumulation of hexoestrol with response t o bilateral adrenalectomy and oophorectomy. Lancet 2:796 --779. Feherty, P., Farrer-Brown, G., Kellie, A. E. (1971). Estradiol receptors in carcinoma and benign disease of the breast: An in vitro assay. Br. J. Cancer 25:697-710. Gross, S. J. (1970). Specificities of steroid antibodies in steroid determinations. In “Immunologic Methods in Steroid Determinations.” F. G. Peron and B. V. Caldwell (Eds.). New York: Appleton-Century-Crofts Education Division Meridith Corporation, pp. 41 -62. Hotchkiss, J . , Atkinson, L. E., and Knobil, E. (1971). Serum estrogen and leuteinizing hormone concentrations during the menstrual cycle of rehesus monkey. Endocrinol. 89: 177-183. Jensen, E. V., DeSombre, E. R., and Jungblut, P. W. (1967). Estrogen receptors in hormone responsive tissues and tumors. In “Endogenous Factors Influencing Host-Tumor Balance” R. W. Witsler, T. L. Dao, and S. Wood, Jr., (Eds.). Chicago: University of Chicago Press, pp. 15-68.
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Jensen, E. V., Block, G. E., Smith, S., Kyser, K. and DeSombre, E. R. (1971). Estrogen receptors and breast cancer response to adrenalectomy. In “Prediction of Response in Cancer Therapy,” T. C. Hall (Ed.). Nat. Cancer Inst. Monog. 34:55-70. Jensen, E. V., and DeSombre, E. R. (1973). Estrogen-receptor interaction. Science 182: 126-134. Korenman, S. G., and Dukes, B. A. (1970). Specific estrogen binding by the cytoplasm of human breast carcinoma. J. Clin. Endocrinol. Metab. 30:639-645. Leung, B. S., Manaugh, L. C., and Wood, D. C. (1973). Estradiol receptors in benign and malignant disease of the breast. Clin. Chm. Acta. 66:69-76. Mass, H., Engel, B., Hochmeister, H., Lehmenn, F., and Trams, G.(1972). Estrogen receptors in human breast cancer tissue. Am. J. Obstet. Gynecol. 113:377-382. McGuire, W. L., and Julian, J. A. (1971). Comparison of macromolecular binding of estradiol in hormonedependent rat mammary carcinoma. Cancer Res. 31: 1440-1450. McGuire, W. L., Huff, K., Jennings, A., and Chamness, G. C. (1972). Mammary carcinoma: A specific biochemical defect in autonomous tumors. Science 175:335-336. McGuire, W. L., and Chamness, G. C. (1973). Studies on the estrogen receptor in breast cancer. In “Receptors for Reproductive Hormones,” B. W. O’MaUey and A. R. Means (Eds.). New York, Plenum Press, pp. 113-136. McGuire, W. L. (1973). Estrogen receptors in human breast cancer. J. Clin. Inves. 52:73-77. McGuire, W. L., Chamness, G. C., Costlow, H. E., and Shephered, R. E. (1974). Hormone dependence in breast cancer. Metabolism 23:15-100. Mobbs, B. G. (1966). The uptake of tritiated estradiol by dimethylbenzanthracene-induced mammary tumors of the rat. J. Endocrinol. 36:409-414. Savlov, E. D., Wittliff, J . L., Hilf, R., and Hall, T. C. (1974). Correlations between certain biochemical properties of breast cancer and response to therapy: A preliminary report. Cancer 33: 303-309. Wittliff, J. L., Hilf, R., Brooks, W. F., Jr., Savlov, E. D., Hall, F. C., and Orlando, R. A. (1972). Specific estrogen-binding capacity of the cytoplasmic receptor in normal and neoplastic breast tissues of humans. Cancer Res. 32:1983-1992.
Estrogen and Estrogen Receptors of Breast .......................................................................................... .......................................................................................... VELAYUDAN SAHADEVAN, M.D., Ph.D., CHARLES P. PERLIA, M.D., STEVEN G. ECONOMOU, M.D., and HOWARD SKY-PECK, Ph.D. Human breast cancer can be divided into a group that contains specific receptor sites for estrogen and a group without such specific estrogen-binding sites. The presznce of specific estrogen receptors in some tumors indicating hormonal dependency has been shown t o be of predictive value for endocrine treatment. This would greatly improve therapeutic planning for patients with breast cancer. Tumor tissue from 52 patients was investigated for content of both cytosol estrogen and estrogen receptor. I n addition, the total tumor estrogen was also determined in 14 of these tumors. The results of this investigation show two distinct groups: one group containing both estrogen receptor and estrogen and a second group with n o receptor but with measurable amount of estrogen. Tumors with estrogen receptors have higher tissue levels of estrogen than tumors without specific estrogen receptor. Even in the absence of estrogen receptor, however, most tumor tissue examined contained a measurable amount of estrogen.
........................................................................................... ......................................................................................... KEY WORDS: breast cancer, estrogen, estrogen receptor, estrogen antiserum, total tumor estrogen, cytosol estrogen
INTRODUCTION When tritium labeled estradiol was injected into patients with metastatic breast cancer prior to adrenalectomy, the cancer of patients responding to adrenalectomy was found to concentrate radioactive estrogen more intensively than the metastatic tissue of nonresponding patients (Folca et al., 1961). This was followed by the observation that dimethylbenzanthracene (DMBA)-induced hormone-dependent rat mammary tumors had higher in vitro uptake of radioactive estrogen that did autonomous tumors (Mobbs, 1966). It was further demonstrated that the preferential uptake of estrogen by hormone-dependent breast tumors could be inhibited by synthetic analogous of estrogen (Jensen et al., 1967). Subsequently, it was shown that the uptake of estrogen by cancer tissue depended on the presence of a specific estrogen-binding protein in the cytoplasm of such tissue (Jensen et al., 1967; Mobbs 1966). This high affinity of estrogen-binding protein is known as Section of Medical Oncology, Department of Medicine, Department of General Surgery and Department of Biological Chemistry, Presbyterian-St. Luke’s Hospital, Chicago, Iliinois. Address all correspondence t o Dr. V. Sahadevan, Presbyterian-St. Luke’s Hospital, 175 3 West Congress Parkway, Chicago, Illinois 60612.
467 0 197.5 A l a n R . Liss, I n c . , 1.50 Fifth A v e n u e , New York, N . Y . 10011
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estrogen receptor. The physical characteristics of this estrophilic protein are shown to be an 8s and/or 4s protein (Jensen and DeSombre, 1973; McCuire and Chamness (1973). When breast tumor tissue contains estrogen-receptor protein, the tumor nucleus is also known to accumulate estrogen (McGuire and Julian, 1971). Nuclei from tumors that are devoid of estrophilic cytoplasmic receptors do not have the ability to concentrate estrogen. On the other hand, if such tumors are incubated with estrogen receptors of uterus, the tumor nuclear binding of estrogen can be readily established (McGuire et al., 1972). It is obvious therefore, that the nucleus of tumor tissue that does not contain estrogen receptor can be induced t o bind estrogen in the presence of exogenous estrogen-receptor protein. Human breast tumors show a wide range of estrogen-receptor proteins (McGuire 1973; Korenman and Dukes, 1970; Feherty et al., 1971; Wittliff et al., 1972; DeSombre et al., 1974). It is reported that up to about 50% of breast tumors have measurable amounts of estrogen-receptor protein (DeSombre et al., 1974). The frequency of estrogenreceptor proteins in tumors of postmenopausal patients is considerably higher than in tumors of premenopausal patients (DeSombre et al., 1974). Usually the receptor concentration of the tumor does not decrease during the progression of the disease. Occasionally, however, the metastasis of the same patient may be with and without estrogen receptor (DeSombre et al., 1974). Tumors that are shown to have estrogen-receptor sites are known to respond to such endocrine treatments as oophorectomy, adreanalectomy, or additive endocrine therapy (Jensen et al., 1971; McGuire et al., 1974). The rate of response to endocrine treatment of patients with tumors that contained estrogen receptors was about 77%. The response rate was about 3% in patients with tumors devoid of estrogen receptor. (Jensen et al., 1971). Similar reports on tumor regression and its relationship to estrogen receptor have been reported by several other independent study groups (Savlov et al., 1974; Mass et al., 1972; Braunsberg et al., 1973). It has been suggested that the variation of receptor concentrations in tumors may be related to tumor contents of epithelial and nonepithelial cells (McCuire et al., 1974). No correlation, however, is found between the histology of a tumor and its estrogen receptor concentration (Wittliff et al., 1972). An alternative hypothesis is that in the presence of endogenous tumor estradiol only a few unoccupied receptor sites would be available for the binding of added exogenous radioactive estrogen (McGuire et al., 1974). It is important, therefore, t o determine the tissue levels of estrogen of tumors with and without estrogen receptor. Saturation of all estrogen receptors with endogenous estrogen may give false negative results. The investigations reported in this est rogen-receptor assay paper were undertaken to assess the relationship of estrogen receptor and tissue levels of estrogen of tumors with and without estrogen receptors. MATERIALS AND METHODS Patient Selection Surgical specimens were obtained from patients referred to the Presbyterian-St. Luke’s Hospital for the management of primary or metastatic breast cancer. Both estrogenreceptor and tumor-estrogen determinations were made on tissue obtained at the time of corrective mastectomy or when it was obtained to confirm recurrence of disease. The patients were evaluated and followed in close cooperation between the surgeon and medical oncologist.
Estrogen and Estrogen Receptors of Breast Cancer
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Radioimmunoassay of Tumor Tissue Contents of Estrogen Antiserum to estradiol-17 beta. Conjugate of bovine serum albumin with estradiol17 beta was prepared by carbodiimide reaction (Gross, 1970). This congugate, together with the complete Freund’s adjuvant and physiologic saline, was used for the immunization of sheep. After several subcutaneous injections of this antigen prepared at weekly intervals, blood was drawn and the antiserum tested for its ability t o bind with radioactive estradiol 17 beta. At an effective dilution of 1:200,000 this antiserum binds about 50% of the added radioactive estradiol-17 beta. Crossreaction of estradiol-17 beta antiserum with other related steroids. The antiserum against estradiol-17 beta cross-reacts with estrone almost with the same affinity as the estradiol-17 beta. At an effective antiserum dilution of 1:200,000, almost identical binding of both estradiol-17 beta and estrone are observed. The magnitude of crossreaction of this antiserum with the estriol was significantly less than with estrone. Crossreaction with the steroid is observed only when the antiserum is diluted to 1:40,000. At this dilution, the antibody affinity to estrone is nearly the same as for estradiol-17 beta. As shown in Fig. 2 , at a higher antiserum dilution of 1 :200,000 its cross-reaction is much less. No cross-reaction of this antiserum is observed with other steroids (Fig. 1). Since no attempts were made to separate the various components of estrogen from tumor tissue extract, the values obtained for tumor estrogen in this study represent the total tissue levels of estrogen. The related antibody affinity to various steroids is shown in Figs. 1 and 2. Extraction of estrogen. The tumor cytosol was prepared as described in the section on estrogen-receptor assay. The cytosol contents of estrogen was extracted with 3 volumes of ethanol. The whole ethanol extraction was evaporated to dryness under a stream of air. The residue was redissolved in phosphate saline buffer prior to radioimmunoassay. The
5
10
20
25
50
lm
2m
5m
1003
PG STEROID
Fig. 1. Standard curve showing the inhibition of 3H-estradiol-17 beta binding to antibody by increments of unlabeled estradiol-17 beta. As with testosterone, all other steroids (cholesterol, progesterone, 1 1-alpha-OH-progesterone, pregnenolone, 19-OH-androstendione, dehydroapepiondrosterone, hydrocortisone, and dehydropregnenolone) failed to bind to this antiserum.
Sahadevan et al.
470
10
5
10
20
25
50
1M
zoo
5m
ICUI
PG HORMONE
Fig. 2. Standard curves showing binding affinity to antibody by various estrogens.
total tissue estrogen was extracted by digesting 100 mg tumor tissue with 0.2 ml 25% HC1 and, after a short period of homogenization, extracting the estrogen twice with 2 ml of ether. The ether extract was evaporated and, prior to radioimmunoassay of estrogen, was redissolved in 0.1 M phosphate saline buffer, pH 7.4. The average estrogen extraction efficiency by these procedures was about 60-70%. Radioimmunoassay procedures Standard curve. Into duplicate siliconized glass tubes, estradiol- 17 beta dissolved in ethanol was added at a range of 2.5 to 500 pg and the ethanol evaporated under a stream of air. Into such tubes there was added 0.1 ml of 1:100,000 antiserum dilution and about 10 pg 3H-estradiol-17 beta in 0.1 ml volume. The dilutions of antiserum and radioactive estradiol were made in phosphate buffer containing 0.1%gelatine as a carrier protein. The tubes were incubated for 12 hr at 4°C. The separation of antibody bound and unbound 3H-estradiol-17beta was done by the addition of 0.1 ml of a 1.5% dextran-coated charcoal in phosphate buffer (Hotchkiss et al., 1971). The tubes were then centrifuged and the supernatant containing the antiserum bound radioactive estrogen measured by liquid scintillation counting. The lowest measurable amount of estradiol- 17 beta by this procedure was found to be 2.5. Estimation of estrogen content of specimens. As in the preparation of the standard curves, to the tubes containing estrogen extracted from the tissue, both antiserum and 3Hestradiol-I7 beta were added after evaporation of its ether contents. After incubation for 12 hr, the bound and unbound radioactive estrogen were separated and the radioactivity measured as described above. Since the percentage of antibody bound estrogen is proportional to the nonradioactive estrogen content of the specimen, the tissue levels of estrogen were calculated from the standard curve and corrections made for the extraction efficiency. The efficiency of the assay system. The recovery of added nonradioactive estrogen to tissue, its extraction, and its subsequent radioimmunoassay were used as parameters to test the efficiency of this assay. As shown in Fig. 3, the recovery, when adjusted to the
Estrogen a n d Estrogen Receptors of Breast Cancer
47 1 50
45
35
0
3
P
25
. . 5
10
. 20
im
50
2m
500
€2
Fig. 3. Recovery of nonradioactive estrogen added to tumor tissue. Increments of nonradioactive estrogen were added to tumor and the estrogen extracted and assayed. When adjusted to extraction efficiency, the recovery experiments run parallel with the standard curves constructed with the same amount of added nonradioactive estrogen.
extraction efficiency, runs parallel with the standard curve constructed with the same amounts of added nonradioactive estrogen. Estrogen receptor assay. Immediately after removal of the cancerous tissue, the specimen was taken to the laboratory and kept on crushed ice. After removing fat, necrotic tumor, and connective tissue, the tumor specimen was either immediately processed for estrogen receptor and estrogen determination or stored frozen. In preparation for tumor cytosol, approximately 0.5-1 gm tumor tissue was pulverized with an automatic frozen tissue pulverizer and subsequently homogenized with the homogenizer PT ST (Brinkman Instruments). Four ml tris-EDTA buffer (0.01 M tris-HC1,0.0015 M EDTA, pH 7.4) was used for 1 gm of tumor tissue for the assay done by the dextran-coated charcoal procedure. Much lesser volume of buffer is used for the assay using sucrose gradient procedures. The homogenate was next centrifuged at 20,000 rpm for 30 min and this supernatant used for the initial dextran-coated charcoal assay. For the sucrose gradient assay procedure, the supernatant was further centrifuged at 150,000 g for 45 min in a Beckman Ultracentrifuge L5-65. During this processing the tissue was kept at about 0°C. Preliminary assay of estrogen receptor by dextran-coated charcoal procedure. The principle of this assay procedure consists of estimation of tumor cytosol bound radioactive estradiol in the presence and absence of either nonradioactive estrogen or its analogue U11, 100 (Nafoxidine) and subsequent separation of bound and unbound Hestradiol by dextran-coated charcoal (Korenman and Dukes, 1970; Leung et al., 1973).
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Because of the molecular similarity of Nafoxidine to estrogen, this compound competes with estrogen for the binding site of receptor. The competitive inhibition of 3H-estradiol binding to receptor protein by Nafoxidine will decrease the estrogen receptor bound radioactivity if the specimen contains specific estrogen receptor. The percentage inhbition of radioactive estrogen binding by Nafoxidine is calculated from the difference in the receptor bound radioactivity in the presence and absence of Nafoxidine. To duplicate tubes, 0.5 ml aliquots of cytosol were added to a mixture of 50 pl of Tris-EDTA buffer with or without 10 p1 of lop4 M Nafoxidine. After 15 min incubation at 0°C 10 pg of 3H-estradiol were added and the mixture incubated at 0°C for an additional 30 min. After incubation the bound and unbound radioactive estrogen was separated with 0.1 ml of a suspension containing 1.5 gm Norite charcoal and 150 mg of Dextran 70/100 ml Tris-EDTA buffer. After adding the charcoal the mixture was incubated for 10 min, the unbound estradiol was adsorbed by the charcoal and then separated by centrifugation. The radioactivity of the bound H-estradiol containing supernatant was measured by liquid scintillation countings in a Packard Automatic Tri-Carb Liquid Scintillation Spectrometer System, model 3330. The specific binding of estrogen by its receptor protein was calculated from the difference in the receptor bound radioactivity in the presence and absence of Nafoxidine. The receptor concentration is expressed as femtomoles (lo-'' moles) 3H-estradiol bound/gm wet weight of tumor. Estrogen receptor assay by sucrose-gradient analysis. Except for the following changes this assay procedure was essentially the same as described by McGuire (1973). About 150 p1 of tumor cytosol prepared as described above were added to a mixture of 50 pl of Tris-EDTA buffer with or without 10 p1 of lop4 M Nafoxidine. After 15 min of incubation at O"C, 10 pg of 3H-estradiol were added to the mixture and incubated at 0°C for an additional 30 min. After incubation, the unbound 3H-estradiol was removed with 0.1 ml of a 1.5% solution of dextran-coated charcoal and subsequent centrifugation to remove the charcoal. Approximately 200 pl of the supernatant were layered onto a cold linear 5-20% sucrose gradient that was prepared in the same buffer as the cytosol. The gradient was then centrifuged at 44,000 rpm for 15 hr in a Beckman ultracentrifuge L5-65. After centrifugation, 0.3 ml fractions were collected by puncturing the bottom of the tube, and the distribution of radioactivity was measured by liquid scintillation countings. The specific estrogen-receptor binding of H-estradiol was identified by isotopic profiles from sucrose gradient and differences in the bound radioactivity at the 8s or 4s region in the presence and absence of Nafoxidine. Figure 4 illustrates the specific estrogen-receptor binding of H-estradiol and its inhibition by both Nafoxidine and nonradioactive estradiol. RESULTS Tumor Estrogen All 52 tumor specimens were examined for their cytosol estrogen concentration. Sufficient tumor tissue from only 14 patients was available for the estimation of total tissue contents of estrogen. Tumors of postmenopausal patients in general had lower tumor estrogen, particularly if they did not have estrogen receptor. Among the 20 specimens without estrogen receptor (from postmenopausal patients), 9 had no measurable cytosol estrogen. The average cytosol estrogen level of these patients was 82 pg per gm wet weight of tumor tissue. On
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Fig. 4. Sucrose gradient assay of estrogen receptors. Both Nafoxidine (NAF) and nonradioactive estrogen (E2) inhibits the 3H-estradiol binding. Since this competitive inhibition is observed both at 8s region (bottom of the tube) and at 4s (top of the tube) this tumor specimen contains both 8s and 4s receptor protein.
the other hand, of the 19 with estrogen receptor from postmenopausal patients, only 5 of the tumor cytosols were devoid of estrogen. The remaining 14 had a relatively higher cytosol estrogen level; the average was 135 pg per gm wet weight of tumor. The tumor cytosol contents of estrogen in premenopausal patients was in general much higher even if these tumors were negative for estrogen receptor. The tumors of 10 premenopausal patients that were negative for estrogen receptor had an average of 145 pg estrogen per gm tumor tissue. This is nearly twice the cytosol estrogen contents of similar patients with tumors negative for estrogen receptor. In pre- and postmenopausal patients the cytosol estrogen content of tumors with estrogen receptor did not show any significant difference; their average values were 133 and 135 pg per gm wet weight of tumor, respectively. These results are summarized in Tables I and 11. The cytosol content of estrogen, however, may not be a true reflection of total tumor contents of estrogen. A greater part of the estrogen may be bound to the nucleus (McGuire and Julian, 1971). On 14 tumors, both cytosol and total estrogen contents were estimated and their reiationship to estrogen receptor assessed (Table 111). The estrogen contents of tumors of preand postmenopausal patients were significantly different in the presence of estrogen
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Endogenous Tumor Cytosol Estrogen of Postmenopausal Patients
TABLE I.
Receptor Positive No. 1 2 3 4 5 6
I 8 9 10 11 12 13 14 15 16 17 18 19
Patient Ro Kr Hu cog
La Ma HaC Col He Sa Qu Ke HaH Ta Jo ThE Ku BeJ WoA
Average
Receptor f moles/gm
Receptor Negative Estrogen pg/g
No.
Patient We Kil Kiz SchM
88 71 4 24 57 174 I06 315 45 178 112 232 22 22 58 19 40 59 372 656
96 245 0 0 137 490 158 34 0 25 123 62 78 0 0 57 196 508 323
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
164
135
Average
AnP
SchR Wi Fo Oa Ar
WOE Go ThI: Ad AnE BeV Wh Pe Mu
Estrogen pg/gm
0 0 7 0 10 11 0 0 4 1 0 10 0 0 0 0 0 0 12
239 0 0 20 0 193 435 0 0 0 372 45 100 0 17 59 23 0 0
3
82
Endogenous Tumor Cytosol Estrogen of Premenopausal Patients
TABLE 11.
Receptor Positive Receptor
No.
Patient
f moles/gm
1 2 3
Jo Dr LuD
318 88 270
225
Average
Receptor f moles/gm
Receptor Negative Estrogen pg/gm
Receptor
Estrogen pg/gm
No.
Patient
f moles/gm
52 347 0
1 2 3 4 5 6 7 8 9 10
Ge McB LuN Ba La Pi Sk SchN Br zy
0 0 0 0 0 3 0 0 0 17
209 56 51 173 50 476 103 0 0 335
133
Average
2
145
receptor than when this specific receptor protein was absent. Tumors with estrogen receptors had higher estrogen levels both in tumor cytosol and in total tumor tissue. In the absence of estrogen receptor, however, there was significant estrogen in tumor tissue, and this was much higher in specimens which were taken from premenopausal patients and which did not contain estrogen receptor.
47 5
Estrogen and Estrogen Receptors of Breast Cancer
TABLE 111.
Endogenous Tumor Estrogen and Estrogen Receptor
Estrogen Receptor Positive
No.
1 2 3 4
Patient
Estrogen Receptor Negative
Estrogen Pgk
Receptor f moles/gm of tissue
Cytosol
Total
No.
232 40 656 88
123 57 323 347
652 99 1267 371
1 2 3 4 5 6 7 8 9 10
254
213
597
Qu ThE WoA Dr
Average
Estrogen pg/gm
Patient
Receptor f moles/gm of tissue
Cytosol
Total
Ki SchM Ad AnE BeV Wh No Ge* Sk SchN*
7 0 0 0 0 0 9 0 0 0
0 20 0 17 59 23 148 209 103 0
12 169 263 0 89 175 771 232 303 284
2
58
230
Average
*Premenopausal
Estrogen Receptor Both estrogen receptor and the estrogen content of tumor were investigated in 52 patients. Estrogen-receptor values over 20 f moles per gm wet weight of tumor tissue are arbitrarily taken as positive for estrogen receptor and values below this are considered as negative for estrogen receptor. This value was taken to account for the technical errors involved in the present system of estimations of estrogen receptors. Tumor tissue was positive for estrogen receptor in 22 of the 52 patients. When these patients were grouped according to their menstrual status, there was a distinct difference in the occurrence of estrogen-receptor activity in the tumors of postmenopausal patients. Among the 13 premenopausal patients, in only 3 did the tumor show estrogen receptor. Their estrogen-receptor contents varied from 88 to 318 f moles per gin tumor tissue. On the other hand, among the 39 tumor tissues obtained from the postmenopausal patients, 19 had tumors with estrogen receptor ranging from 22 to 656 f moles. These results are summarized in Tables I and 11.
DISCUSSION As already mentioned, there is an increased recognition of the significance of estrogen receptors in breast tumors. Knowledge of these receptors would greatly improve the ability to plan therapy for patients with breast cancer. The significance, however, of a wide range of receptor concentration in tumors with estrogen receptor is not known. It has been suggested that the endogenous tumor estradiol contents may significantly alter the tumor receptor concentration (McGuire et al., 1974). If all the estrogen-receptor sites of the tumor tissue are occupied by endogenous estrogen, no additional binding of the added radioactive estrogen to the tumor cytosols could be observed. With our present methods of estrogen-receptor assay, only the binding of added radioactive estrogen to tumor cytosol is measured and, therefore, the saturation of receptor protein with
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Sahadevan et al.
endogenous hormones may be highly significant in the evaluation of tumor containing estrogen receptor. In a study of 6 patients, it was shown that the endogenous tumor estrogen may not saturate all the binding sites of estrogen receptor (Korenman and Dukes, 1970). In the study reported here, a more sensitive radioimmunoassay for determination of tumor estrogen is employed. The relationship of endogenous tumor estrogen concentration to tumor estrogen receptor is assessed in tumor tissue of 5 2 patients. This study demonstrates that the contents of estrogen in tumor tissue is related to both endogenous estrogen and the tumor content of estrogen receptor. In postmenopausal patients, the tumor estrogen content is markedly enhanced in the presence of estrogen receptor. On the other hand, in tumors of premonopausal women, the presence of estrogen receptor seems not to have an influence on the tumor content of estrogen. This may indicate that the estrogen content of tumor is mostly influenced by the endogenous estrogen. More studies on premenopausal tumors, however, are required for further analysis of this observation. On the other hand, in postmenopausal patients the presence of estrogen receptor is closely associated with a higher endogenous tumor estrogen concentration. This may indicate that in the presence of estrogen receptor, the estrogen utilization by the tumor tissue is enhanced. In conclusion, this study clearly demonstrates that in those tumors with estrogen receptor the wide range of estrogen receptor is unrelated to the endogenous tumor content of estrogen. Contrary to previous speculation, with a higher estrogen receptor in a tumor there is proportionately higher estrogen content. This may be related to the rapid turnover of estrogen in tumors with estrogen receptor. The estrogen binding and the transformation of the estrogen receptor from its 8s to 5s receptor may be very rapid in tumors with estrogen receptor.
ACKNOWLEDGMENTS This work was supported by the Breast Cancer Research Foundation and Public Health Service Grant #4729 1. The authors greatly acknowedge the excellent technica1 assistance of Mrs. Jean Harrison, Mrs. Victoria Sahadevan, and Miss Sirima Silva. REFERENCES Braunsberg, H., James, V. H. T., and Irvine, W. T. (1973). Prognostic significance of’ estrogen uptake by human breast cancer tissue. Lancet 1:163-165. DeSombre, E. R., Smith, S., Block, G. E., Fergusin, D. J., and Jensen, E. V. (1974). Prediction of breast cancer response t o endocrine therapy. Cancer Chemother. Rep. 58:s 13-519. Folca, P. J., Glascock, R. F., and Irvine, W. T. (1961). Studies with Tritium-labeled hexoestrol in advanced breast cancer. Comparison of tissue accumulation of hexoestrol with response t o bilateral adrenalectomy and oophorectomy. Lancet 2:796 --779. Feherty, P., Farrer-Brown, G., Kellie, A. E. (1971). Estradiol receptors in carcinoma and benign disease of the breast: An in vitro assay. Br. J. Cancer 25:697-710. Gross, S. J. (1970). Specificities of steroid antibodies in steroid determinations. In “Immunologic Methods in Steroid Determinations.” F. G. Peron and B. V. Caldwell (Eds.). New York: Appleton-Century-Crofts Education Division Meridith Corporation, pp. 41 -62. Hotchkiss, J . , Atkinson, L. E., and Knobil, E. (1971). Serum estrogen and leuteinizing hormone concentrations during the menstrual cycle of rehesus monkey. Endocrinol. 89: 177-183. Jensen, E. V., DeSombre, E. R., and Jungblut, P. W. (1967). Estrogen receptors in hormone responsive tissues and tumors. In “Endogenous Factors Influencing Host-Tumor Balance” R. W. Witsler, T. L. Dao, and S. Wood, Jr., (Eds.). Chicago: University of Chicago Press, pp. 15-68.
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Jensen, E. V., Block, G. E., Smith, S., Kyser, K. and DeSombre, E. R. (1971). Estrogen receptors and breast cancer response to adrenalectomy. In “Prediction of Response in Cancer Therapy,” T. C. Hall (Ed.). Nat. Cancer Inst. Monog. 34:55-70. Jensen, E. V., and DeSombre, E. R. (1973). Estrogen-receptor interaction. Science 182: 126-134. Korenman, S. G., and Dukes, B. A. (1970). Specific estrogen binding by the cytoplasm of human breast carcinoma. J. Clin. Endocrinol. Metab. 30:639-645. Leung, B. S., Manaugh, L. C., and Wood, D. C. (1973). Estradiol receptors in benign and malignant disease of the breast. Clin. Chm. Acta. 66:69-76. Mass, H., Engel, B., Hochmeister, H., Lehmenn, F., and Trams, G.(1972). Estrogen receptors in human breast cancer tissue. Am. J. Obstet. Gynecol. 113:377-382. McGuire, W. L., and Julian, J. A. (1971). Comparison of macromolecular binding of estradiol in hormonedependent rat mammary carcinoma. Cancer Res. 31: 1440-1450. McGuire, W. L., Huff, K., Jennings, A., and Chamness, G. C. (1972). Mammary carcinoma: A specific biochemical defect in autonomous tumors. Science 175:335-336. McGuire, W. L., and Chamness, G. C. (1973). Studies on the estrogen receptor in breast cancer. In “Receptors for Reproductive Hormones,” B. W. O’MaUey and A. R. Means (Eds.). New York, Plenum Press, pp. 113-136. McGuire, W. L. (1973). Estrogen receptors in human breast cancer. J. Clin. Inves. 52:73-77. McGuire, W. L., Chamness, G. C., Costlow, H. E., and Shephered, R. E. (1974). Hormone dependence in breast cancer. Metabolism 23:15-100. Mobbs, B. G. (1966). The uptake of tritiated estradiol by dimethylbenzanthracene-induced mammary tumors of the rat. J. Endocrinol. 36:409-414. Savlov, E. D., Wittliff, J . L., Hilf, R., and Hall, T. C. (1974). Correlations between certain biochemical properties of breast cancer and response to therapy: A preliminary report. Cancer 33: 303-309. Wittliff, J. L., Hilf, R., Brooks, W. F., Jr., Savlov, E. D., Hall, F. C., and Orlando, R. A. (1972). Specific estrogen-binding capacity of the cytoplasmic receptor in normal and neoplastic breast tissues of humans. Cancer Res. 32:1983-1992.
