Journal of Surgical Oncology 9:595-601 (1977)
Estrogen Receptor Levels in Hormonally Progressive Mammary Tumors .......................................................................................... ..........................................................................................
DONALD G . BRONN, M.s., UNTAE KIM, M.D.*, LORETTA DIVECCHIA, M.s., WILLIAM E. KlBBEY, M.D., and JOHN P. MINTON, M.D., Ph.D. Tumor samples from 4 different stages of hormonal progrcssion in the MT-W9 series of rat mammary tumors were analyzed for estrogen receptors by the deutran-coated charcoal method in order to further explore the clinical implications of the cstrogen rcceptor assay. The findings indicate that although the presence of estrogen receptors is not an exclusive characteristic of hormonal dependency. thcir absence is indicative of a later stage of hormonal progression which might be of more immediate clinical consequence because hornionally autonomous tumors have faster growth rates than hormonally dependent tumors. I t is also suggested that it may be necessary to initiate chemotherapy as an adjuvant to endocrine therapy for metastatic breast cancer patients with hormonally dependent tumors in order to avoid the eventual proliferation of hormonally autonomous tumor cells which are present in hornionally dependent tumors.
..... .....
...... ....
Key words: estrogen receptor, MT-W9 mammary tumors, hormonal progression
It has been well known since the beginning of this century (Beatson. 1896) that breast cancer can be either hornionally dependent or independent as evidenced by the fact that some recurrent breast cancers regress following endocrine ablation (oophorectomy, adrenalectomy , or hypophysectomy). Such regression occurs in approximately one third of all breast cancer patients so treated (MacDonald, 1962; Taylor, 1962). Unfortunately, for the remaining two thirds, endocrine ablation is totally ineffective in bringing about any change in the course of the disease. Thus, in order t o spare the trauma of surgery t o those breast cancer patients whose tumors would not respond t o endocrine ablation, an effort was made t o identify beforehand which breast tumors were hornionally responsive and which were not. The method which has had the most success in accomplishing this goal has been the estrogen receptor (ER) assay. The application of this assay to breast cancer patients came about as a result of the demonstration that the clinical response t o honnone therapy could be correlated with the presence or absence of an estrogen-specific binding protein (estrogen receptor) in the cytoplasm of breast cancer cells (Jensen et al., 1971). Thus, it became possible to characterize breast tumors as being hornionally dependent (responsive)
From the Department of Surgery. The Ohio Statc University College of Medicine, Columbus, Ohio, and *Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York Address reprint requcsts to Dr. John P. Minton, Department of Surgery, The Ohio State University, 410 W. Tenth Avenue, Columbus, OH 43210.
59 5
0 1977 Alan R . Liss, Inc., 1 5 0 Fifth A v e n u e , New York, N Y 10011
596
Bronn et al.
in the presence of estrogen receptors and hormonally independent (autonomous) in the absence of estrogen receptors. Expanded use of the estrogen receptor assay in recent years has resulted in the finding that approximately 92% of all breast cancer patients with a negative ER value d o not respond to endocrine therapy. It was also found that only 55% of breast cancer patients with positive E R values responded favorably t o endocrine therapy (McCuire et al., 1975). Thus, although the E R assay was most effective in determining which breast cancer patients should not undergo endocrine therapy by a negative E R finding, it was only 55% accurate in determining which breast cancer patients should undergo endocrine therapy, by a positive E R finding. The development of the MT-W9 series of mammary tumors in rats (Kim and Depowski, 1975) provides an experimental model for human mammary adenocarcinoma in varying stages of hormonal responsiveness. These tumors were originally induced with a subcarcinogenic dose of 3-methylcholanthrene (MCA) plus an extraneous source of mammotropin in high levels provided by a mammotropic pituitary tumor present in the same animal host. This resulted in the development of the MT-W9 mammary tumor which is a mammotropin-dependent hormonally responsive tumor. This tumor would not have manifested itself clinically in response to the subcarcinogenic dose of MCA unless high levels o f mammotropin were provided. Consequently, any subtle decrease in hormone levels causes immediate regression. This MT-W9 population eventually gave rise t o an estrogen-dependent hormonally responsive tumor population, the MT-W9A, which is no longer as sensitive t o subtle changes in hormone levels as its antecedent. However, if endocrine ablation is performed, completely removing the source of estrogen, this tumor will also promptly regress. Subsequently, the MT-W9A tumor population produced the fully autonomous MT-W9B which grows well in all syngeneic rats regardless of their hormonal status and is totally unaffected by any endocrine ablative procedure. From this hormonally autonomous tumor population a fourth tumor cell line was identified, the MT-W9D. This tumor was classified as an advanced hormonally autonomous mammary tumor since it arose from a tumor population which was already honnonally autonomous. Similarly, it is completely unaffected by any endocrine ablative procedure. The hormonal and cytogenetic characteristics of each of these stem cell lines have been stable for over a decade. It was, therefore, the aim of this study t o examine the estrogen receptor levels in the MT-W9 series of mammary tumors at different stages of hormonal dependency in order t o further explore the clinical implications of the ER assay.
MATERIALS AND METHODS A total of 18 mammary tumor samples were excised from tumor-bearing Wistar/ Furth female rats. The mammary tumors borne by these rats were of the MT-W9 series and collectively comprise 4 stages of hormonal progression: mammotropin-dependent hormonally responsive (W9). estrogen-dependent liormonally responsive (W9A), hormonally autonomous (W9B), and advanced hormonally autonomous (W9D). After excision, the specimens were immersed directly in liquid nitrogen within 30 seconds and kept in a liquid nitrogen storage tank at -196°C until analyses were performed. Frozen tissues were pulverized with a steel mortar and pestle after which time 250300 mg of the pulverulent was homogenized manually in 2 ml of buffer A (cold TrisHCl buffer containing 10 mM Tris-HC1, 1.5 mM EDTA, and 0.5 mM dithiothreitol, pH
Estrogen Receptors and Hormonal Progression
597
7.4) with a Ten Broeck glass homogenizer on ice using 20 strokes. Then an additional 2 ml of buffer A was added and mixed with 5 strokes. The homogenate was then centrifuged at 100,000 X g for 60 minutes in a Beckman refrigerated ultracentrifuge (Model L3-40). The supernatant was removed without disturbing the lipid layer at the t o p of the tube. This supernatant fraction (cytosol) was kept on ice until the binding assay was performed. Estradiol-17/3-6,7-' H (54.3 Ci/niM, New England Nuclear, Boston, Mass.) was diluted with benzene-ethanol solution (9:l) until a 25 ng/nil solution was obtained (stock solution). This solution was prepared every 3 months and stored in a refrigerator at 4°C. One milliliter o f stock solution was dried with a stream of nitrogen, and 1 ml o f absolute ethanol was added. The ethanol solution was then diluted with 24 ml o f buffer A t o make a 1 ng/ml solution. Two hundred microliters of cytosol were incubated with 100 1.11of 'H-estradiol at concentrations of 5, 10, 25, 50. and 100 picograms (0.018 t o 0.367 picomoles) in duplicate for 18 hours at 4°C. The solutions o f 'H-estradiol of various concentrations were prepared from the 1 ng/ml solution within 1 hour before incubation. Two hundred microliters of buffer A were also incubated with graduated amounts of 'H-estradiol in triplicate for control and blank. At least 2 ml of cytosol were needed per assay. The dextran-coated charcoal method of Korenman (1968) was used with minor modifications; in these assays the dextran-coated charcoal suspension was prepared using 500 mg of Norit A and 50 mg of Dextran T-70 in 100 ml of buffer A. After 18 hours of incubation, 0.5 nd of charcoal-dextran suspension was added t o and mixed in each of the tubes containing cytosol solution and those containing blank solution. To each of the control tubes, 0.5 ml of buffer A was added. After incubation for 15 minutes at 4"C, each tube was centrifuged for 10 minutes at 2,100 X g. Three hundred microliters of supernatant of each of the tubes were added t o 10 ml of Bray's solution (naphthalene 60 g, PPO 4 g, POPOP 0.2 g, methanol 100 ml, and ethylene glycol 20 ml in 1 liter of p-dioxane) in a scintillation vial (Bray, 1960). The tritium count was measured in a lipid scintillation couriter (Searle Delta 300). Hormone receptor binding in each o f the test tubes was calculated from the counts o f the assay tubes and the counts of the blank and control tubes. Scatchard's plot (Scatchard, 1949) was made with a ratio of bound t o free estradiol and the amount of bound estradiol per ml cytosol. From this chart, the dissociation constants and estrogen-binding capacities (estrogen receptor values) were obtained. The dissociation constants were derived by dividing the abscissa intercept by the ordinate intercept. The cytosol protein level was determined by the method of Lowry et al. (1951). The estrogen receptor values in fenitomoles/nig cytosol protein were then determined by dividing the abscissa intercept by the Lowry protein value.
RESULTS Six different runs of the estrogen receptor assay were done. Twelve of the total 18 samples analyzed were from homionally autonomous (independent) mammary tumors, and only 6 were from hormonally dependent (responsive) tumors. Specific nstrogenbinding capacity (positive estrogen receptor values) was found in 12 of the 18 samples analyzed (66.6%). The high affinity binding specificity o f the estrogen receptor assay was confirmed b y the dissociation constants (Leclercq e t al., 1973). Positive estrogen receptor values were found in all 6 of the hormonally dependent tumors (10070). However, 6 of the 12 homionally autonomous tumors were also found t o have positive estrogen receptor
598
Bronn et al.
values (50%). Therefore, although twice as many liornionally autononious tumors than hormonally dependent tumors were assayed the majority of tumors still had positive estrogen receptor values (Fig. 1). The estrogen receptor values in femtomoles/nig cytosol protein for the various tumor stages showed that the W9 tumors had positive ER values ranging from 40 t o 49 femtomoles. The W9A tumors had positive ER values ranging from 20 t o 5 3 ferntomoles and the W9B tiimors had positive ER values ranging from 14 t o 38 ferntomoles. However, 3 of the 8 samples of the W9B tumor stage had negative ER values (37.5%). The W9D tumors had 1 positive ER value of 37 ferntomoles whereas 3 of the 4 samples (75%) analyzed in this stage were found t o be negative for estrogen receptor values (Table I). The hormonally dependent tumors had positive ER values which ranged from 20 t o 59 ferntomoles and the hormonally autononiom tumors had positive ER values which ranged from 14 to 38 ferntomoles.
DISCUSSION The acceptance of the estrogen receptor assay as a tool for predicting response t o endocrine therapy in breast cancer patients has resulted in the categorization of mammary tumors as being either estrogen receptor positive or estrogen receptor negative. Since 92% of breast cancer patients with a negative ER finding do not respond t o endocrine therapy, this would indicate that the ER assay is an accurate method for de t e rin ini ng which mammary t LI mo rs are ho rnio ti ally auto ti onio us. However , only 5 55%) of breast cancer patients with a positive ER finding respond favorably t o endocrine therapy (McGuire et al., 1975). This would indicate that 45% of mammary tumors which have estrogen receptors are nevertheless hormonally autononious.
18
ANALYZED TUMOR POSITIVE ESTROGEN RECEPTOR VALUES
8 6
3I-
& 3 0
z 0 OVERALL (66.6Ok)
HORMONALLY H m N / \ L L Y DEPEAUTCNDMOUS (IOOok) ( SOo/o)
1;ig. 1. Comparison of the iiuniber of positive I.'K findings t o thc nutither of samples analyzed. €lorinonally dependent tumors (W9 and W9A) and the Iiormonally autonomous tumors (W9H and W9D) are also compared separately. T h e difference between the number of samples analyzed and the n u m b e ~ of positive ER findings is t h e number of negative EK determinations.
Estrogen Receptors and Hormonal Progression
599
TABLE I. Estrogen Receptor Values (in femtomoles/mg cytosol protein) of the Rat Mammary Tumor Samples for the 4 Different Stages of Hormonal Progression in the MT-W9 Series RUN
W9
W9A
W9B
20 55 53
14 38
49 40 59
No. of Samplcs 5% Ncg
3 0
N eg Neg Neg
22 25 35
3 0
8 31.5
W9D
Neg Neg N eg
31
4 75
Similarly, the presence o f estrogen receptors in 50% of the hornionally autonomous mammary tumors of the MT-W9 series (W9B and W9D) further underscores the fact that estrogen receptors are not an exclusive characteristic of hormonal dependency. It also suggests a qualitative difference in estrogen receptors. Therefore. mammary tumors should more properly be divided into 3 categories: hormonally dependent with estrogen receptors, hormonally autonomous without estrogen receptors, and hormonally autonomous with estrogen receptors. It is the hornionally autonomous tumors with estrogen receptors that are the cause of the 45% false positive rate of the ER assay in breast cancer patients. Furthermore, since the hornionally autonomous tumors exhibited ER levels as high as 38 femtomoles/mg cytosol protein whereas the hormonally dependent tumors had ER levels as low as 20 femtomoles/nig cytosol protein, the hormonal dependency of a mammary tumor may not be entirely related t o the quantity of ER present. Since the estrogen receptor assay is only a quantitative determination of the estrogen receptors present, an ER assay of the cytosol is incapable of qualitatively distinguishing between the functional estrogen receptors of the hornionally dependent tumors and the nonfunctional estrogen receptors of the hormonally autonomous tumors. The nonfunctionality of estrogen receptors, despite their presence in the cytosol, may be the result of the inability of the E R complex to translocate into the nucleus, or perhaps the ER coniplex is unable t o bind t o its acceptor sites on the chromatin. Negative ER findings in the MT-W9 series, which were found only in the hornionally autonomous tumors, indicate that there is a corresponding absence of estrogen receptors with the progression of the tumors toward hormonal autonomy. This is further emphasized by the higher incidence of negative ER findings in the advanced hormonally autonomous stage (W9D). It can, therefore, be concluded that once a mammary tumor cell population is negative for estrogen receptors, it will remain negative because a negative ER finding is also indicative of a later stage of hormonal progression which appears t o be an irreversible process.
600
Bronn et al.
The presence of estrogen receptors in the hormonally autonomous tumors also identifies these tumors as being in an intermediate stage between hormonal dependence and absolute autonomy. Although mammary tumors at this intermediate stage are hormonally independent, they nevertheless produce remnant estrogen binding proteins which represent the hormonal past of the tumor cell. As progression to hormonal autonomy proceeds, subsequent cell populations are altered to such an extent that they no longer produce an estrophilic protein. Thus, qualitative changes in the receptors may precede their ultimate absence in the hormonally autonomous tumor cells. The more rapid growth rates of the hormonally autonomous tumors over those of the hormonally dependent tumors of the MT-W9 series further suggest that a negative ER finding is also indicative of a highly proliferative tumor population. This may be of more immediate clinical consequence than the slower growing hornionally dependent tumors. Chromosomal analyses of the MT-W9 series of mammary tumors indicated that all tumors contained both hormonally dependent and autonomous cell clones, and plurality of one over the other seems to determine whether a tumor is hormonally dependent or autonomous (Kim and Depowski, 1975). This suggests that the immediate failure of endocrine therapy in breast cancer patients, initiated as a result of a positive ER assay, is due to the dominance of the hormonally autonomous tumor cells possessing estrogen receptors. However, the eventual failure of initially successful endocrine therapy would be caused by the elimination of the hormonally dependent tumor population which results in the proliferation of the hormonally autonomous tumor cells. An ER assay of the subsequent tumor may not necessarily show any substantial decrease in ER levels since hormonally autonomous tumors often possess estrogen receptors. Therefore, it may be necessary to initiate chemotherapy as an adjuvant to endocrine therapy for breast cancer patients with hormonally dependent tumors in order to avoid the subsequent proliferation of hormonally-autonomous tumor cells.
ACKNOWLEDGMENTS
This investigation was supported by Grant No. 5R25CA18016 and Contract No. 1-CB-23864 from the National Cancer Institute, DHEW, by The Ohio State University Development Fund No. 522809, and by contributions from Phi Beta Psi Sorority, and The Ohio Chapter of The Grand Order of the Eastern Star.
REFERENCES Beatson, G.T. (1896). On the treatment of inoperable cases of carcinoma of the mammary. Lancet 2: 104- 107. Bray, G.A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. Jensen, E.V., Block, G.E., Smith, S., Kyser, K., and DeSombre, E.R. (1971). Estrogen receptors and breast cancer response t o adrenalectomy. Natl. Cancer Inst. Monograph 34:55-79. Kim, U., and Depowski, M.J. (1975). Progression from hormone dependence to autonomy in mammary tumors as an in vivo manifestation of sequential clonal selection. Cancer Res. 35:2068 2077.
Estrogen Receptors and Hormonal Progression
60 1
Korenman, S.G. (1968). Radio-ligand binding assay of specific estrogens using a soluble uterine macromolecule. J. Clin. Endocrinol. Metab. 28:127- 132. Leclercq, G., Heuson, J.C., Schoenfeld, R., Matthelem, W.H., and Tagnon, H.J. (1973). Estrogen receptors in human breast cancer. Eur. J. Cancer 9:665-673. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 193265-275. MacDonald, I. (1962). Endocrine ablation in disseminated mammary carcinoma. Surg. Gynecol. Obstet. 115:215-222. McGuire, W.L., Carbone, P.P., Sears, M.E., and Escher, G.C. (1975). Estrogen receptors in human breast cancer: An overview. In “Estrogen Receptors in Human Breast Cancer,” W. L. McGuire, P.P. Carbone, and E.P. Vollmer (eds). New York: Raven Press, p. 1-7. Scatchard, G. (1949). The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51 1660-672. Taylor, S.G. (1962). Endocrine ablation in disseminated mammary carcinoma. Surg. Gynecol. Obstet. 1 15 :44 3-448.
Estrogen Receptor Levels in Hormonally Progressive Mammary Tumors .......................................................................................... ..........................................................................................
DONALD G . BRONN, M.s., UNTAE KIM, M.D.*, LORETTA DIVECCHIA, M.s., WILLIAM E. KlBBEY, M.D., and JOHN P. MINTON, M.D., Ph.D. Tumor samples from 4 different stages of hormonal progrcssion in the MT-W9 series of rat mammary tumors were analyzed for estrogen receptors by the deutran-coated charcoal method in order to further explore the clinical implications of the cstrogen rcceptor assay. The findings indicate that although the presence of estrogen receptors is not an exclusive characteristic of hormonal dependency. thcir absence is indicative of a later stage of hormonal progression which might be of more immediate clinical consequence because hornionally autonomous tumors have faster growth rates than hormonally dependent tumors. I t is also suggested that it may be necessary to initiate chemotherapy as an adjuvant to endocrine therapy for metastatic breast cancer patients with hormonally dependent tumors in order to avoid the eventual proliferation of hormonally autonomous tumor cells which are present in hornionally dependent tumors.
..... .....
...... ....
Key words: estrogen receptor, MT-W9 mammary tumors, hormonal progression
It has been well known since the beginning of this century (Beatson. 1896) that breast cancer can be either hornionally dependent or independent as evidenced by the fact that some recurrent breast cancers regress following endocrine ablation (oophorectomy, adrenalectomy , or hypophysectomy). Such regression occurs in approximately one third of all breast cancer patients so treated (MacDonald, 1962; Taylor, 1962). Unfortunately, for the remaining two thirds, endocrine ablation is totally ineffective in bringing about any change in the course of the disease. Thus, in order t o spare the trauma of surgery t o those breast cancer patients whose tumors would not respond t o endocrine ablation, an effort was made t o identify beforehand which breast tumors were hornionally responsive and which were not. The method which has had the most success in accomplishing this goal has been the estrogen receptor (ER) assay. The application of this assay to breast cancer patients came about as a result of the demonstration that the clinical response t o honnone therapy could be correlated with the presence or absence of an estrogen-specific binding protein (estrogen receptor) in the cytoplasm of breast cancer cells (Jensen et al., 1971). Thus, it became possible to characterize breast tumors as being hornionally dependent (responsive)
From the Department of Surgery. The Ohio Statc University College of Medicine, Columbus, Ohio, and *Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York Address reprint requcsts to Dr. John P. Minton, Department of Surgery, The Ohio State University, 410 W. Tenth Avenue, Columbus, OH 43210.
59 5
0 1977 Alan R . Liss, Inc., 1 5 0 Fifth A v e n u e , New York, N Y 10011
596
Bronn et al.
in the presence of estrogen receptors and hormonally independent (autonomous) in the absence of estrogen receptors. Expanded use of the estrogen receptor assay in recent years has resulted in the finding that approximately 92% of all breast cancer patients with a negative ER value d o not respond to endocrine therapy. It was also found that only 55% of breast cancer patients with positive E R values responded favorably t o endocrine therapy (McCuire et al., 1975). Thus, although the E R assay was most effective in determining which breast cancer patients should not undergo endocrine therapy by a negative E R finding, it was only 55% accurate in determining which breast cancer patients should undergo endocrine therapy, by a positive E R finding. The development of the MT-W9 series of mammary tumors in rats (Kim and Depowski, 1975) provides an experimental model for human mammary adenocarcinoma in varying stages of hormonal responsiveness. These tumors were originally induced with a subcarcinogenic dose of 3-methylcholanthrene (MCA) plus an extraneous source of mammotropin in high levels provided by a mammotropic pituitary tumor present in the same animal host. This resulted in the development of the MT-W9 mammary tumor which is a mammotropin-dependent hormonally responsive tumor. This tumor would not have manifested itself clinically in response to the subcarcinogenic dose of MCA unless high levels o f mammotropin were provided. Consequently, any subtle decrease in hormone levels causes immediate regression. This MT-W9 population eventually gave rise t o an estrogen-dependent hormonally responsive tumor population, the MT-W9A, which is no longer as sensitive t o subtle changes in hormone levels as its antecedent. However, if endocrine ablation is performed, completely removing the source of estrogen, this tumor will also promptly regress. Subsequently, the MT-W9A tumor population produced the fully autonomous MT-W9B which grows well in all syngeneic rats regardless of their hormonal status and is totally unaffected by any endocrine ablative procedure. From this hormonally autonomous tumor population a fourth tumor cell line was identified, the MT-W9D. This tumor was classified as an advanced hormonally autonomous mammary tumor since it arose from a tumor population which was already honnonally autonomous. Similarly, it is completely unaffected by any endocrine ablative procedure. The hormonal and cytogenetic characteristics of each of these stem cell lines have been stable for over a decade. It was, therefore, the aim of this study t o examine the estrogen receptor levels in the MT-W9 series of mammary tumors at different stages of hormonal dependency in order t o further explore the clinical implications of the ER assay.
MATERIALS AND METHODS A total of 18 mammary tumor samples were excised from tumor-bearing Wistar/ Furth female rats. The mammary tumors borne by these rats were of the MT-W9 series and collectively comprise 4 stages of hormonal progression: mammotropin-dependent hormonally responsive (W9). estrogen-dependent liormonally responsive (W9A), hormonally autonomous (W9B), and advanced hormonally autonomous (W9D). After excision, the specimens were immersed directly in liquid nitrogen within 30 seconds and kept in a liquid nitrogen storage tank at -196°C until analyses were performed. Frozen tissues were pulverized with a steel mortar and pestle after which time 250300 mg of the pulverulent was homogenized manually in 2 ml of buffer A (cold TrisHCl buffer containing 10 mM Tris-HC1, 1.5 mM EDTA, and 0.5 mM dithiothreitol, pH
Estrogen Receptors and Hormonal Progression
597
7.4) with a Ten Broeck glass homogenizer on ice using 20 strokes. Then an additional 2 ml of buffer A was added and mixed with 5 strokes. The homogenate was then centrifuged at 100,000 X g for 60 minutes in a Beckman refrigerated ultracentrifuge (Model L3-40). The supernatant was removed without disturbing the lipid layer at the t o p of the tube. This supernatant fraction (cytosol) was kept on ice until the binding assay was performed. Estradiol-17/3-6,7-' H (54.3 Ci/niM, New England Nuclear, Boston, Mass.) was diluted with benzene-ethanol solution (9:l) until a 25 ng/nil solution was obtained (stock solution). This solution was prepared every 3 months and stored in a refrigerator at 4°C. One milliliter o f stock solution was dried with a stream of nitrogen, and 1 ml o f absolute ethanol was added. The ethanol solution was then diluted with 24 ml o f buffer A t o make a 1 ng/ml solution. Two hundred microliters of cytosol were incubated with 100 1.11of 'H-estradiol at concentrations of 5, 10, 25, 50. and 100 picograms (0.018 t o 0.367 picomoles) in duplicate for 18 hours at 4°C. The solutions o f 'H-estradiol of various concentrations were prepared from the 1 ng/ml solution within 1 hour before incubation. Two hundred microliters of buffer A were also incubated with graduated amounts of 'H-estradiol in triplicate for control and blank. At least 2 ml of cytosol were needed per assay. The dextran-coated charcoal method of Korenman (1968) was used with minor modifications; in these assays the dextran-coated charcoal suspension was prepared using 500 mg of Norit A and 50 mg of Dextran T-70 in 100 ml of buffer A. After 18 hours of incubation, 0.5 nd of charcoal-dextran suspension was added t o and mixed in each of the tubes containing cytosol solution and those containing blank solution. To each of the control tubes, 0.5 ml of buffer A was added. After incubation for 15 minutes at 4"C, each tube was centrifuged for 10 minutes at 2,100 X g. Three hundred microliters of supernatant of each of the tubes were added t o 10 ml of Bray's solution (naphthalene 60 g, PPO 4 g, POPOP 0.2 g, methanol 100 ml, and ethylene glycol 20 ml in 1 liter of p-dioxane) in a scintillation vial (Bray, 1960). The tritium count was measured in a lipid scintillation couriter (Searle Delta 300). Hormone receptor binding in each o f the test tubes was calculated from the counts o f the assay tubes and the counts of the blank and control tubes. Scatchard's plot (Scatchard, 1949) was made with a ratio of bound t o free estradiol and the amount of bound estradiol per ml cytosol. From this chart, the dissociation constants and estrogen-binding capacities (estrogen receptor values) were obtained. The dissociation constants were derived by dividing the abscissa intercept by the ordinate intercept. The cytosol protein level was determined by the method of Lowry et al. (1951). The estrogen receptor values in fenitomoles/nig cytosol protein were then determined by dividing the abscissa intercept by the Lowry protein value.
RESULTS Six different runs of the estrogen receptor assay were done. Twelve of the total 18 samples analyzed were from homionally autonomous (independent) mammary tumors, and only 6 were from hormonally dependent (responsive) tumors. Specific nstrogenbinding capacity (positive estrogen receptor values) was found in 12 of the 18 samples analyzed (66.6%). The high affinity binding specificity o f the estrogen receptor assay was confirmed b y the dissociation constants (Leclercq e t al., 1973). Positive estrogen receptor values were found in all 6 of the hormonally dependent tumors (10070). However, 6 of the 12 homionally autonomous tumors were also found t o have positive estrogen receptor
598
Bronn et al.
values (50%). Therefore, although twice as many liornionally autononious tumors than hormonally dependent tumors were assayed the majority of tumors still had positive estrogen receptor values (Fig. 1). The estrogen receptor values in femtomoles/nig cytosol protein for the various tumor stages showed that the W9 tumors had positive ER values ranging from 40 t o 49 femtomoles. The W9A tumors had positive ER values ranging from 20 t o 5 3 ferntomoles and the W9B tiimors had positive ER values ranging from 14 t o 38 ferntomoles. However, 3 of the 8 samples of the W9B tumor stage had negative ER values (37.5%). The W9D tumors had 1 positive ER value of 37 ferntomoles whereas 3 of the 4 samples (75%) analyzed in this stage were found t o be negative for estrogen receptor values (Table I). The hormonally dependent tumors had positive ER values which ranged from 20 t o 59 ferntomoles and the hormonally autononiom tumors had positive ER values which ranged from 14 to 38 ferntomoles.
DISCUSSION The acceptance of the estrogen receptor assay as a tool for predicting response t o endocrine therapy in breast cancer patients has resulted in the categorization of mammary tumors as being either estrogen receptor positive or estrogen receptor negative. Since 92% of breast cancer patients with a negative ER finding do not respond t o endocrine therapy, this would indicate that the ER assay is an accurate method for de t e rin ini ng which mammary t LI mo rs are ho rnio ti ally auto ti onio us. However , only 5 55%) of breast cancer patients with a positive ER finding respond favorably t o endocrine therapy (McGuire et al., 1975). This would indicate that 45% of mammary tumors which have estrogen receptors are nevertheless hormonally autononious.
18
ANALYZED TUMOR POSITIVE ESTROGEN RECEPTOR VALUES
8 6
3I-
& 3 0
z 0 OVERALL (66.6Ok)
HORMONALLY H m N / \ L L Y DEPEAUTCNDMOUS (IOOok) ( SOo/o)
1;ig. 1. Comparison of the iiuniber of positive I.'K findings t o thc nutither of samples analyzed. €lorinonally dependent tumors (W9 and W9A) and the Iiormonally autonomous tumors (W9H and W9D) are also compared separately. T h e difference between the number of samples analyzed and the n u m b e ~ of positive ER findings is t h e number of negative EK determinations.
Estrogen Receptors and Hormonal Progression
599
TABLE I. Estrogen Receptor Values (in femtomoles/mg cytosol protein) of the Rat Mammary Tumor Samples for the 4 Different Stages of Hormonal Progression in the MT-W9 Series RUN
W9
W9A
W9B
20 55 53
14 38
49 40 59
No. of Samplcs 5% Ncg
3 0
N eg Neg Neg
22 25 35
3 0
8 31.5
W9D
Neg Neg N eg
31
4 75
Similarly, the presence o f estrogen receptors in 50% of the hornionally autonomous mammary tumors of the MT-W9 series (W9B and W9D) further underscores the fact that estrogen receptors are not an exclusive characteristic of hormonal dependency. It also suggests a qualitative difference in estrogen receptors. Therefore. mammary tumors should more properly be divided into 3 categories: hormonally dependent with estrogen receptors, hormonally autonomous without estrogen receptors, and hormonally autonomous with estrogen receptors. It is the hornionally autonomous tumors with estrogen receptors that are the cause of the 45% false positive rate of the ER assay in breast cancer patients. Furthermore, since the hornionally autonomous tumors exhibited ER levels as high as 38 femtomoles/mg cytosol protein whereas the hormonally dependent tumors had ER levels as low as 20 femtomoles/nig cytosol protein, the hormonal dependency of a mammary tumor may not be entirely related t o the quantity of ER present. Since the estrogen receptor assay is only a quantitative determination of the estrogen receptors present, an ER assay of the cytosol is incapable of qualitatively distinguishing between the functional estrogen receptors of the hornionally dependent tumors and the nonfunctional estrogen receptors of the hormonally autonomous tumors. The nonfunctionality of estrogen receptors, despite their presence in the cytosol, may be the result of the inability of the E R complex to translocate into the nucleus, or perhaps the ER coniplex is unable t o bind t o its acceptor sites on the chromatin. Negative ER findings in the MT-W9 series, which were found only in the hornionally autonomous tumors, indicate that there is a corresponding absence of estrogen receptors with the progression of the tumors toward hormonal autonomy. This is further emphasized by the higher incidence of negative ER findings in the advanced hormonally autonomous stage (W9D). It can, therefore, be concluded that once a mammary tumor cell population is negative for estrogen receptors, it will remain negative because a negative ER finding is also indicative of a later stage of hormonal progression which appears t o be an irreversible process.
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Bronn et al.
The presence of estrogen receptors in the hormonally autonomous tumors also identifies these tumors as being in an intermediate stage between hormonal dependence and absolute autonomy. Although mammary tumors at this intermediate stage are hormonally independent, they nevertheless produce remnant estrogen binding proteins which represent the hormonal past of the tumor cell. As progression to hormonal autonomy proceeds, subsequent cell populations are altered to such an extent that they no longer produce an estrophilic protein. Thus, qualitative changes in the receptors may precede their ultimate absence in the hormonally autonomous tumor cells. The more rapid growth rates of the hormonally autonomous tumors over those of the hormonally dependent tumors of the MT-W9 series further suggest that a negative ER finding is also indicative of a highly proliferative tumor population. This may be of more immediate clinical consequence than the slower growing hornionally dependent tumors. Chromosomal analyses of the MT-W9 series of mammary tumors indicated that all tumors contained both hormonally dependent and autonomous cell clones, and plurality of one over the other seems to determine whether a tumor is hormonally dependent or autonomous (Kim and Depowski, 1975). This suggests that the immediate failure of endocrine therapy in breast cancer patients, initiated as a result of a positive ER assay, is due to the dominance of the hormonally autonomous tumor cells possessing estrogen receptors. However, the eventual failure of initially successful endocrine therapy would be caused by the elimination of the hormonally dependent tumor population which results in the proliferation of the hormonally autonomous tumor cells. An ER assay of the subsequent tumor may not necessarily show any substantial decrease in ER levels since hormonally autonomous tumors often possess estrogen receptors. Therefore, it may be necessary to initiate chemotherapy as an adjuvant to endocrine therapy for breast cancer patients with hormonally dependent tumors in order to avoid the subsequent proliferation of hormonally-autonomous tumor cells.
ACKNOWLEDGMENTS
This investigation was supported by Grant No. 5R25CA18016 and Contract No. 1-CB-23864 from the National Cancer Institute, DHEW, by The Ohio State University Development Fund No. 522809, and by contributions from Phi Beta Psi Sorority, and The Ohio Chapter of The Grand Order of the Eastern Star.
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