Robert Paridaens

Aromatase, 17β-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normal glandular breast tissue in postmenopausal women

In a study of the origin of estrogens in patients with breast cancer, the concentrations of estrogens and their androgen precursors, and aromatase and 17 beta-hydroxysteroid dehydrogenase (E2DH) activities were determined in normal glandular and cancerous breast tissue. The correlation between tissue estrogens, precursor concentrations, enzyme activities and plasma levels and/or receptor status were calculated. In both normal glandular and carcinomatous breast tissue, the concentrations of androstenedione (A), dehydroepiandrosterone (DHEA), 5 androstene-3 beta, 17 beta-diol (5-Adiol), estrone (E1), estradiol (E2) and progesterone (P) were significantly higher than plasma concentrations. While testosterone (T) concentrations were similar, dehydroepiandrosterone (DHCA) and estrone sulphate (E1S) concentrations were lower in tissue than in plasma. In carcinomatous tissue androgen concentrations were lower, but estrogen concentrations were higher than in glandular breast tissue. Estradiol (E2) concentration was positively correlated with the receptor concentration with the mean E2 concentration corresponding to an estimated receptor occupancy of about 25%, probably sufficient for a submaximal biological response. Aromatase and E2DH (E2----E1) activities were observed in all breast cancer and glandular breast tissues, activities being higher in carcinoma than in glandular breast tissues; nevertheless, aromatase activity accounts probably only for a small fraction of tissue estrogen concentration. E2DH, but not aromatase activity, was significantly higher in estrogen receptor positive than in estrogen receptor negative tissues and was negatively correlated with tissue dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) concentration; the latter two steroids are non competitive inhibitors of E2DH which inactivates E2 to E1. This effect of DHEA(S) may constitute a mechanism by which these androgens stimulate cancer growth and a rationale (besides suppression of estrogen precursors) for medical or surgical adrenalectomy in hormone sensitive metastatic mammary cancer. E2DH activity might constitute an additional marker of hormone dependency of mammary cancer.

Clinical significance of the quantitative assessment of estrogen receptors in advanced breast cancer

The predictive value of the estrogen receptor (ER) assay with regard to the response to hormonal treatment was analyzed in women with advanced breast carcinoma. The significance of ten clinical variables of putative prognostic value was also investigated. A total of 49 courses of endocrine therapy were available for study. The respective merits of using the receptor information as a qualitative or a quantitative variable were compared. Linear logistic regression analysis showed that the quantitative information was significantly related to the therapeutic response (P < 0.0001) and proved to be superior to the qualitative information. Compared with the clinical variables tested with the logistic model, receptor concentration was by far the most important single predictor of response. Nevertheless, introduction of two of these clinical variables (i.e., age and menopausal status) into the model in addition to receptor concentration improved its predictive value. Presented in graphic form, the improved model provides a simple means to estimate the probability that a given patient will respond to endocrine therapy.

Correlation between nuclear cytomorphometric parameters and estrogen receptor levels in breast cancer

The authors studied the relationships existing between various cytomorphonuclear parameters recorded on 25 primary breast cancers and their estrogen receptor (ER) content. Cell image analyses of Feulgenstained imprint smears, allowing determination of morphologic, densitometric, as well as textural parameters, were assessed by using the SAMBA 200 system (TITN, France). The ER levels were measured by the conventional dextran‐coated charcoal assay. The authors then divided the 25 cancers into three categories: (1) “ER‐negative or poorly positive tumors,” i.e., those having less than 50 fmol ER/mg protein; (2) “ER‐positive tumors,” i.e., those containing between 50 and 150 fmol ER/mg protein; and (3) “ER highly positive tumors,” i.e., those having more than 150 fmol ER/mg protein. The authors results show that ER‐negative or poorly positive breast cancers possess cells with bigger nuclei and higher DNA content, related to higher proliferation index than ER‐rich tumors. Furthermore, the chromatin pattern of cells from ER‐negative or poorly positive breast cancers is significantly more condensed than the thinly textured chromatin of ER highly positive tumors. Cell image analysis of Feulgen‐stained imprints is proposed as an additional tool for grading malignancy.

Steroid dynamics in the normal and carcinomatous mammary gland

UNLABELLEDnAs an approach to the study of the origin of estrogens in breast cancer tissue, the concentration of estrogens and their androgen precursors, as well as aromatase and 17 beta-dehydrogenase activities in normal glandular (GL) and cancerous (CA) breast tissue were determined and correlations with plasma levels and/or receptor status were studied. In both normal GL and CA breast tissue, steroid concentrations were significantly higher than plasma conc., except for dehydroepiandrosterone sulphate (DHEAS), estrone sulphate (E1S) and testosterone (T). Androgen conc. were lower, but estrogen conc. were higher in CA than in GL breast tissue. Estradiol (E2) conc. was positively correlated with the E2R conc., mean E2 conc. corresponding to an estimated E2R occupancy of about 25%. Aromatase and 17 beta-hydroxysteroid dehydrogenase (E2DH) (E2----E1) activities were observed in all breast CA and GL tissues, aromatase accounting probably only for a small fraction of tissue estrogens. E2DH, but not aromatase activity, was significantly higher in E2R+ than in E2R- tissues and was negatively correlated with tissue dehydroepiandrosterone (DHEA) and DHEAS conc.; the latter two steroids are non competitive inhibitors of E2DH which inactivates E2 to E1.nnnCONCLUSIONnin both normal and carcinomatous breast tissue, conc. of E1 and E2 are significantly higher than in plasma, suggesting either uptake or local synthesis. As to the latter, aromatase activity accounts probably only for a minor fraction of the tissue estrogens. Breast CA tissue has higher aromatase and E2DH activity than normal glandular tissue, E2 conc. and E2DH activity being higher in E2R+ hormone sensitive, tumors than in E2R-tumors. Tissue conc. of DHEA(S) which inhibits oxidative inactivation of E2, is negatively correlated with E2DH activity and may have an important modulating role in intratissular estrogen metabolism.

Inhibitory action of androstenedione on the proliferation and cell cycle kinetics of aromatase-free MXT and MCF-7 mammary tumour cell lines.

The effects of androstenedione (AD) on cell proliferation and kinetics have been measured in MXT mouse and MCF-7 human mammary cancer cell lines using SAMBA 200 cell image analysis of Feulgen-stained nuclei. At a concentration of 0.01 microM AD inhibited the proliferation of both cell lines whereas a higher dose (1 microM) was inhibitory on MCF-7 cell proliferation but stimulatory in MXT cells. It is unlikely that these effects are due to aromization of AD into oestrogen since (a) both cell lines were devoid of aromatase and (b) both cell lines were similarly affected by oestradiol (E2), being stimulated at low concentrations and inhibited at high doses. Furthermore, inhibition by AD seems to occur, at least in part, by blockade of the cell cycle whereas that by E2 appears to be cell cycle independent.

Effect of ovariectomy, hypophysectomy and/or GnRH analog (HRF) administration on the cell proliferation of the MXT mouse hormone-dependent mammary tumor

The MXT tumor is an experimental mammary neoplasm which is maintained by serial transplantation using B6D2F1 mice, and which contains significant amounts of estrogen and progesterone receptors. The aim of the present study is to examine the effects of ovariectomy (OVX) or ovariectomy plus hypophysectomy (OVX-HX) on both the macroscopic growth and the cell proliferation of this tumor. This cell proliferation was evaluated by means of in vivo tritiated thymidine autoradiography. In addition, we investigated the effects of a GnRH analog (Gonadorelin: HRF, 5-oxo-Pro-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-hydrochloride) on MTX tumor cell proliferation on 7 day-OVX and 5 day-HX (OVX-HX) mice. The uterine luminal epithelium was chosen to monitor the methodology. Our data clearly demonstrate that there is a delay in the growth of MXT tumors grafted into hypophysectomized animals and, to a lesser degree, ovariectomized animals. With respect to proliferation, castration induced a dramatic decrease of the thymidine labelling index (TLI) in the tissue used to monitor the methodology (the uterine luminal epithemium); in contrast, no cell proliferation was induced by hypophysectomy or HRF administration. In 4-week-old MXT tumors, ovariectomy also markedly decreased the TLI within a few days of its taking place. However hypophysectomy, performed on castrated animals, induced a significant and transient increase of cell proliferation in this neoplasm, an increase which lasted from the 2nd to the 5th day following the operation. The high basal level of MXT cell proliferation recorded in OVX-HX animals decreased dramatically after the administration of HRF between 12 and 48 h prior to the sacrifice of the animals. It is concluded that the HRF exerts a direct effect on the MXT tumor cells, and this HRF might be essential for their growth.

Endocrine effects of trilostane: in vitro and in vivo studies

Trilostane (4-alpha-5-epoxy-17 beta-hydroxy-3-oxo-5-alpha-androstan-2-carbonitrile) is a modified steroidal molecule. In vitro and in vivo studies in rats have shown that it inhibits adrenal, ovarian and placental steroid synthesis. It seems to act by exerting a selective blockade on 3 beta-hydroxysteroid dehydrogenase. In this study, we investigated whether this molecule interacts with hormone receptors for estrogen, androgen or progesterone. We also tried to demonstrate the effect which Trilostane may have on cellular cultures of human mammary carcinoma (MCF-7 Evsa-T). We also studied hormonal modifications in a series of 12 patients treated with different doses of Trilostane, since this drug is supposed to inhibit the production by the adrenal glands of mineralocorticoids, of glucocorticoids and of the precursors of estrogens. Our results indicate that Trilostane does not react with any of the main hormonal sex steroid receptors, nor does it interfere with cultures of human mammary cancer cells either containing estrogen receptors and therefore allegedly hormone-dependent (MCF-7 line), or estrogen receptor-negative cells, presumably independent of hormonal manipulations (Evsa-T cell line). Finally, endocrine studies on postmenopausal women with advanced breast cancer show that Trilostane significantly reduces the plasma levels of estrone and of its major androgen precursor (androstenedione). However, the latter inhibition is no different from that exerted by hydrocortisone acetate administered alone at a dose of 40 mg/day. The results of clinical trials comparing hydrocortisone alone with hydrocortisone plus Trilostane are awaited.

Influence of Fetal Calf Serum in Combination with Pharmacological Doses of Progesterone or Estradiol on Proliferation and Cell Cycle Kinetics of Cultured Mammary Cancer Cells

We describe an original method to monitor clonal cell density (hyperplasia) and the cell cycle kinetics of neoplastic cells simultaneously. We thus characterize the in vitro influence of two different types of fetal calf serum (FCS), defined as FCS-S and FCS-I, on the progesterone- or estradiol-induced effect on proliferation and cell cycle kinetic parameters of the MXT mouse and MCF-7 human mammary cancer cell lines. The two sera were treated with dextran-coated charcoal. The FCS-S serum showed a stimulatory influence on MXT and MCF-7 growth, whereas FCS-I was devoid of any clear-cut influence. The cells were cultured on glass coverslips, placed in Petri dishes containing either a control or a hormone-added medium, fixed for histology and submitted to the Feulgen reaction, which allows selective and quantitative (stoichiometric) staining of DNA. Proliferation and cell cycle kinetics were analyzed on the same sample of cells by means of the SAMBA 200 cell image processor. Our results show that steroid-mediated effects were dramatically modulated according to the type of serum used. Furthermore, they also show that pharmacological doses of progesterone or estradiol decrease MXT cell growth by at least two different mechanisms: the first is related to cell cycle kinetics, i.e. an inhibition of the cells into the S phase, while the second remains unknown but seems to be cell cycle independent. High dose estradiol, but not progesterone, induced the same inhibitory influence on the MCF-7 cells.

Characterization and Therapeutic use of Hormone Dependency of Breast Cancer

SummaryOur work, centered on hormone dependency hreast cancer, has therapeutic and fundament al implications in two main domains:1. Characterization of hormonal responsiveness by receptor assays:Among clinical, biochemical and pathological data, estrogen receptors (ER) are the best barkers of responsiveness to hormonal treatments. The quantitative assay is particularly informative, and a model linking ER concentration to the probability of response has been created, nich guides the clinician for the treatment of indual patients. It is also helpful for the Prospective study of new treatments on groups of Patients.2. Use of interactions between hormones and Wotoxic drugsOn a murine model, it was shown that one single pulse of estradiol exerts a transient vmitogenic effect. This manipulation allows, under very stringent conditions, to amplify the an titumioral action of a cycle-active cytotoxic drug.The concept is now under study in a randomized clinical trial in which patients receive chmotherapy preceeded ...

Estrogen Receptor Variations in Neoplastic Tissue During the Course of Disease in Patients with Recurrent Breast Cancer

In recent years, it has been repeatedly demonstrated that estrogen receptor (ER) assays are useful as a therapeutic guide in the treatment of advanced breast cancer. The validity of this tool in predicting the results of endocrine treatments has been confirmed in our laboratory [1, 2] as well as in many others (review article, ref. [3]). Moreover, we found that among several variables known to have a predictive value, receptor concentration was the one most significantly related to the therapeutic response; the presence or absence of ER was of lesser value [1,2].