PART
11. RECEPTORS IN BREASTCANCER
RECEPTORS IN BREAST CANCER: AN INTRODUCTION J . P. Raynaud Centre de Recherches Roussel UCLAF 93230 Romainville, France
Human breast cancer is a field in which fundamental studies on hormonereceptor interactions seem to have a particularly close bearing on problems that impinge on our daily lives today. I t is now a virtually accepted notion that, in the absence of a significant level of cytoplasmic estrogen receptor, breast cancer patients are most unlikely to respond to available hormonal therapy, whether ablative or additive. It would seem that a simple assay of receptor sites in a biopsy can spare the trauma of surgical or chemical castration to many women. One of the aims of the pharmaceutical industry, apart from the ultimate goal of designing new drugs for the cure of disease, is to find methods, whether tests or reagents, for diagnostic purposes. The search for both new drugs and for new methods, however, is based on the development and refinement of experimental models derived from fundamental research. At present, in our laboratories as in many others. considerable attention is being given to the introduction of a simple receptor assay in human breast cancer and to the interpretation of the results obtained. because, regardless of the techniques used by different teams for receptor measurement, only SO-60% of estrogen receptor-positive patients respond to hormonal therapy. Recently, we have perfected an assay for the measurement of progestin-binding sites in animal tissues, which is now being applied, after appropriate controls, to specimens from breast cancer patients. For a long time, a major problem in the-detection of progestin-specific binding in many species was the interference of progesterone binding to specific plasma proteins, for example, transcortin. Due to this interference and also to the lability of the 7s progesterone-receptor complex, it was extremely difficult to detect, let alone measure, progestin-binding sites. It seemed to us that a simple and effective way of disregarding the interference binding was to use a synthetic radioligand, in this instance R SO20 (17.21-dimethyl-19-norpregna-4,9-diene-3,2O-dione), not bound by transcortin and that, due to the slow dissociation rate of the complex formed with progestin receptor, as determined in studies on animal uterus, "stabilizes" this receptor. This approach introduced the notion of the use of synthetic molecules as receptor "tags" not bound by plasma proteins and entirely specific to the receptor corresponding to a single class of hormone. We have, in fact, shown that R SO20 is virtually totally specific for progestin receptor; it competes slightly (13%) for binding to the dexamethasone-labeled glucocorticoid receptor, a result that has been confirmed by other teams, but not for binding to the estrogen, androgen, or mineralocorticoid receptors. At present, we also have available a highly specific tag for the estrogen receptor, R 2858 ( 1 Ip-methoxy-19-nor-17a1.3.3 10)-pregnatrien-20-yn-3,17-diol).that is not bound by sex steroid-binding plasma protein and that is being used in preliminary assays for estrogen receptors in breast tumors. comparatively with estradiol, with fairly interesting results. Regarding the androgen receptor, although our radioligand R 1881 is not bound by SBP, it competes very markedly for binding to the progestin receptor. These molecules are now being used to measure total cytoplasmic binding sites by exchange assays in various tissues (e.g., rabbit uterus and rat and human prostate).
87
88
Annals New York Academy of Sciences
Under the assumption that measurement of cytoplasmic binding sites would be of diagnostic value to the consulting physician, it is necessary that the assay for the hospital laboratory should meet the following requirements. It should be specific, reproducible, sensitive, and, if possible, adaptable for use on a microscale. It should be preferably simple, rapid, and inexpensive. In our opinion, a method that involves measurement of binding sites by a dextran-coated adsorption exchange assay and use of the above-mentioned synthetic ligands meets the above practical requirements more satisfactorily than do sucrose density gradient ultracentrifugation and agar gel electrophoresis. At present, biochemical controls for extrapolation of the progestin receptor assay from estradiol-primed immature rabbit uterus to human breast tissue are being carried out to determine the most suitable conditions of ligand concentration, incubation time, and temperature for a valid single-point assay that ensures that total exchange of endogenous hormone occurs in a time compatible with receptor stability. In collaboration with a medical team from Villejuif Hospital (France), we have examined tissue specimens from more than 200 patients, with a technique that employs estradiol instead of R 2858 (and therefore does not eliminate interference from binding to contaminating plasma proteins). and conditions of exchange for both estradiol and R 5020 extrapolated directly without controls from animal data. The results are, nevertheless, interesting, particularly for 15 patients for whom not only the concentration of binding sites in the tumor, but also that in a tissue sample taken from a precise region that adjoins the nipple were measured after histopathologic examination of the specimen. The receptor level in tumor tissue was generally significantly higher than in control tissue. Except for two cases of infiltration by malignant cells, the progestin receptor level in control tissue was always equal to zero or negligible. Estrogen receptor level in control tissue, however, reached approximately 200 fmol/g of tissue in five of 15 cases. To what extent interference plasma binding is involved is being studied. These results, however, emphasize the aleatory nature of quantitative assessments when the percentage of malignant cells in the specimen is not known. This immediately brings up the point of the heterogeneity of the tumor: assays on different samples from the same tumor have given different results, and the question arises as to whether all or only some malignant cells contain receptor. Furthermore, because no information is available on the possible cyclic variations in receptor content of normal mammary gland cells, the threshold value above which the tumor may be considered receptor positive and the value above which it may be, furthermore, considered clinically hormone dependent are impossible to determine, except by correlation with hormonal therapy. Prior to the establishment of any clinical correlation, however, a full-scale statistical analysis with a highly controlled technique is required to define the sensitivity of the measurement, particularly of a one-point measurement, because the quantity of tissue is often not sufficient for several determinations. Ideally, it should be possible to perform the assays on drills. The validity of the extrapolation of techniques from animal to man is being studied, but the condition that underlies this extrapolation is that hormone mechanisms in animal tissues and in malignant human breast tissue are similar, if not the same. To check this fundamental assumption, new experimental models have to be introduced, and one of the most promising appears to be that of tissue culture, which will enable in vitro analysis of receptor mechanisms directly on human tissue, in particular, malignant cells. Such a model might provide answers to a few important questions. It would eliminate such problems as heterogeneity, permit comparisons in receptor mechanisms between animal and man (transloca-
Raynaud: Receptors in Breast Cancer
89
tion of the complex to the nucleus, binding to a nuclear acceptor site), and would throw some light on what is probably the most vital question, namely: To what extent is the receptor mechanism implicated in malignant growth? Moreover, this model would be ideal to test all of those molecules, which, from screening data on animal studies, have been suspected to exert antihormonal activity, for instance, by virtue of the rapid dissociation rate of the cytoplasmic complex formed and the subsequently impaired response.
11. RECEPTORS IN BREASTCANCER
RECEPTORS IN BREAST CANCER: AN INTRODUCTION J . P. Raynaud Centre de Recherches Roussel UCLAF 93230 Romainville, France
Human breast cancer is a field in which fundamental studies on hormonereceptor interactions seem to have a particularly close bearing on problems that impinge on our daily lives today. I t is now a virtually accepted notion that, in the absence of a significant level of cytoplasmic estrogen receptor, breast cancer patients are most unlikely to respond to available hormonal therapy, whether ablative or additive. It would seem that a simple assay of receptor sites in a biopsy can spare the trauma of surgical or chemical castration to many women. One of the aims of the pharmaceutical industry, apart from the ultimate goal of designing new drugs for the cure of disease, is to find methods, whether tests or reagents, for diagnostic purposes. The search for both new drugs and for new methods, however, is based on the development and refinement of experimental models derived from fundamental research. At present, in our laboratories as in many others. considerable attention is being given to the introduction of a simple receptor assay in human breast cancer and to the interpretation of the results obtained. because, regardless of the techniques used by different teams for receptor measurement, only SO-60% of estrogen receptor-positive patients respond to hormonal therapy. Recently, we have perfected an assay for the measurement of progestin-binding sites in animal tissues, which is now being applied, after appropriate controls, to specimens from breast cancer patients. For a long time, a major problem in the-detection of progestin-specific binding in many species was the interference of progesterone binding to specific plasma proteins, for example, transcortin. Due to this interference and also to the lability of the 7s progesterone-receptor complex, it was extremely difficult to detect, let alone measure, progestin-binding sites. It seemed to us that a simple and effective way of disregarding the interference binding was to use a synthetic radioligand, in this instance R SO20 (17.21-dimethyl-19-norpregna-4,9-diene-3,2O-dione), not bound by transcortin and that, due to the slow dissociation rate of the complex formed with progestin receptor, as determined in studies on animal uterus, "stabilizes" this receptor. This approach introduced the notion of the use of synthetic molecules as receptor "tags" not bound by plasma proteins and entirely specific to the receptor corresponding to a single class of hormone. We have, in fact, shown that R SO20 is virtually totally specific for progestin receptor; it competes slightly (13%) for binding to the dexamethasone-labeled glucocorticoid receptor, a result that has been confirmed by other teams, but not for binding to the estrogen, androgen, or mineralocorticoid receptors. At present, we also have available a highly specific tag for the estrogen receptor, R 2858 ( 1 Ip-methoxy-19-nor-17a1.3.3 10)-pregnatrien-20-yn-3,17-diol).that is not bound by sex steroid-binding plasma protein and that is being used in preliminary assays for estrogen receptors in breast tumors. comparatively with estradiol, with fairly interesting results. Regarding the androgen receptor, although our radioligand R 1881 is not bound by SBP, it competes very markedly for binding to the progestin receptor. These molecules are now being used to measure total cytoplasmic binding sites by exchange assays in various tissues (e.g., rabbit uterus and rat and human prostate).
87
88
Annals New York Academy of Sciences
Under the assumption that measurement of cytoplasmic binding sites would be of diagnostic value to the consulting physician, it is necessary that the assay for the hospital laboratory should meet the following requirements. It should be specific, reproducible, sensitive, and, if possible, adaptable for use on a microscale. It should be preferably simple, rapid, and inexpensive. In our opinion, a method that involves measurement of binding sites by a dextran-coated adsorption exchange assay and use of the above-mentioned synthetic ligands meets the above practical requirements more satisfactorily than do sucrose density gradient ultracentrifugation and agar gel electrophoresis. At present, biochemical controls for extrapolation of the progestin receptor assay from estradiol-primed immature rabbit uterus to human breast tissue are being carried out to determine the most suitable conditions of ligand concentration, incubation time, and temperature for a valid single-point assay that ensures that total exchange of endogenous hormone occurs in a time compatible with receptor stability. In collaboration with a medical team from Villejuif Hospital (France), we have examined tissue specimens from more than 200 patients, with a technique that employs estradiol instead of R 2858 (and therefore does not eliminate interference from binding to contaminating plasma proteins). and conditions of exchange for both estradiol and R 5020 extrapolated directly without controls from animal data. The results are, nevertheless, interesting, particularly for 15 patients for whom not only the concentration of binding sites in the tumor, but also that in a tissue sample taken from a precise region that adjoins the nipple were measured after histopathologic examination of the specimen. The receptor level in tumor tissue was generally significantly higher than in control tissue. Except for two cases of infiltration by malignant cells, the progestin receptor level in control tissue was always equal to zero or negligible. Estrogen receptor level in control tissue, however, reached approximately 200 fmol/g of tissue in five of 15 cases. To what extent interference plasma binding is involved is being studied. These results, however, emphasize the aleatory nature of quantitative assessments when the percentage of malignant cells in the specimen is not known. This immediately brings up the point of the heterogeneity of the tumor: assays on different samples from the same tumor have given different results, and the question arises as to whether all or only some malignant cells contain receptor. Furthermore, because no information is available on the possible cyclic variations in receptor content of normal mammary gland cells, the threshold value above which the tumor may be considered receptor positive and the value above which it may be, furthermore, considered clinically hormone dependent are impossible to determine, except by correlation with hormonal therapy. Prior to the establishment of any clinical correlation, however, a full-scale statistical analysis with a highly controlled technique is required to define the sensitivity of the measurement, particularly of a one-point measurement, because the quantity of tissue is often not sufficient for several determinations. Ideally, it should be possible to perform the assays on drills. The validity of the extrapolation of techniques from animal to man is being studied, but the condition that underlies this extrapolation is that hormone mechanisms in animal tissues and in malignant human breast tissue are similar, if not the same. To check this fundamental assumption, new experimental models have to be introduced, and one of the most promising appears to be that of tissue culture, which will enable in vitro analysis of receptor mechanisms directly on human tissue, in particular, malignant cells. Such a model might provide answers to a few important questions. It would eliminate such problems as heterogeneity, permit comparisons in receptor mechanisms between animal and man (transloca-
Raynaud: Receptors in Breast Cancer
89
tion of the complex to the nucleus, binding to a nuclear acceptor site), and would throw some light on what is probably the most vital question, namely: To what extent is the receptor mechanism implicated in malignant growth? Moreover, this model would be ideal to test all of those molecules, which, from screening data on animal studies, have been suspected to exert antihormonal activity, for instance, by virtue of the rapid dissociation rate of the cytoplasmic complex formed and the subsequently impaired response.
