Assessment of estrogenic recruitment before chemotherapy in advanced breast cancer: a double-blind randomized study. European Organization for Research and Treatment of Cancer Breast Cancer Cooperative Group.

PURPOSE: In the present phase III study, the specific effect of estrogenic recruitment was assessed by comparing two groups of patients with advanced breast cancer receiving either ethinylestradiol (EE2) or placebo (PL) before chemotherapy (CT). PATIENTS AND METHODS: The therapeutic regimen consisted of (1) estrogen suppression by aminoglutethimide (AGL) 1 g/d plus hydrocortisone (HC) 40 mg/d, with surgical castration performed on premenopausal patients; (2) fluorouracil (5-FU) 500 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide (CPA) 500 mg/m2 (FAC) intravenously (IV) every 3 weeks; (3) following randomization, patients were double-blinded to receive either PL or EE2 50 micrograms exactly 24 hours before receiving FAC. All patients had advanced breast cancer presumably sensitive to endocrine therapy (estrogen receptor-positive [ER+] and/or progesterone receptor-positive [PgR+] status) with measurable lesions; none had received prior systemic antineoplastic therapy for metastatic disease; prior adjuvant hormonal therapy (HT) or CT (without anthracyclines) was allowed if interval since completion was longer than 1 year. RESULTS: Among 154 patients treated according to the protocol, tolerance, response rates, time to progression, and median survival duration were identical in the PL and EE2 groups. Only performance status, dominant metastatic site, and menopausal status seemed to influence response (overall response, 64%), with the highest levels of partial remission (PR) and complete remission (CR) being achieved in premenopausal women (CR plus PR, 26% plus 55%) and in those with dominant soft tissue lesions (CR plus PR, 45% plus 28%). CONCLUSION: We conclude that the validity of the hormonal recruitment concept has not yet been established in clinical practice so that this approach remains experimental. The results achieved by combining (near) complete estrogenic suppression and cyclical FAC chemotherapy are not significantly different from those to be expected with the more conventional use of HT followed by CT in presumably hormone-responsive (ER+) patients.

Assessment of estrogenic recruitment before chemotherapy in advanced breast cancer: preliminary results of a double-blind randomized study of the EORTC Breast Cancer Cooperative Group.

We investigated whether estrogenic recruitment could enhance the antitumor effect of chemotherapy in 165 patients with advanced breast cancer, presumably sensitive to hormonal treatments (ER + and/or PgR + lesions). The therapeutic regimen consisted of: (a) estrogenic suppression by aminoglutethimide 1 g/day + hydrocortisone 40 mg/day; surgical castration in premenopausal patients only; (b) FAC (5FU 500 mg/m2; ADM 50 mg/m2; CPA 500 mg/m2) for 3 weeks; (c) following randomization, exactly 24 h prior to chemotherapy, patients had to take 1 tablet of either placebo (PL) or 50 microgram ethinylestradiol (EE2). Tolerance, responses, time to progression and median survival were identical in both groups. Thus, EE2 before chemotherapy did not contribute to the efficacy of this particular therapeutic regimen, which yielded an overall response rate of 64%. We conclude that the validity of the hormonal recruitment concept has not yet been established in clinical practice, so that this approach remains experimental.

Estrogenic and antiestrogenic down-regulation of estrogen receptor levels: evidence for two different mechanisms.

Preincubation of MCF-7 cells with estradiol (E2) produces a decrease of 3H-E2 binding capacity ("processing"); the strong antiestrogen methylhydroxytamoxifen (MHT) is also effective but with a approximately 100 fold lower efficiency. Parallel immunological measurement of estrogen receptor contents of the cells (ER-EIA from Abbott) revealed that the mechanisms by which these ligands operate are not of the same nature. Thus, while E2 produced a loss of the ER peptide, MHT increased it; indicating an accumulation of a non-binding form of the receptor under its treatment. Measurement of the binding capacity of the cells for 3H-ORG 2058 showed a decrease of PgR concentration after pre-incubation with MHT which contrasted with the classical E2-induced increase of the receptor. MHT at relatively low concentrations also antagonised the E2-induced decrease of 3H-E2 binding capacity; this property did not result from a difference in chemical structure between the ligands since bisphenol a weak estrogenic analogue of MHT failed to show a similar antagonistic activity. This property confers to MHT the ability to reduce the efficiency of E2 to induce PgR. Finally, actinomycin D a known antagonist of the E2-induced processing was found to be totally ineffective towards the MHT processing. This clearly confirmed that the term "processing" covers at least two distinct mechanisms.

Six-year results of a multimodality treatment strategy for locally advanced breast cancer.

Between 1976 and 1982, 59 patients with locally advanced breast cancer were treated with preoperative supervoltage radiotherapy, adjuvant preoperative and postoperative hormonochemotherapy, and modified radical mastectomy. Systemic treatment, which was started simultaneously with radiotherapy, consisted of a combination of daily oral tamoxifen and a monthly alternation of Doxorubicin + vincristine and cyclophosphamide + methotrexate + 5-fluorouracil (CMF). One of each cycle was given preoperatively at half dosage and five of each were repeated postoperatively at full dosage. All patients became operable. Results of pathologic examination of the operative specimen, available in 51 patients, showed complete disappearance of tumor tissue in breast areas in eight patients, of which three still had positive axillary nodes. After a median follow-up time of 6 years locoregional failure was observed in 12 patients (20%) but in only three (5%) did it occur before distant failure. The actuarial median survival of the entire patient population is close to 4 years. Seven patients are alive without recurrence at greater than 9 years. This aggressive multidisciplinary treatment approach is associated with a projected 30% long-term survival (10 years), excellent local control, but substantial toxicity.

Synergism or antagonism between estradiol and progesterone on the cell kinetic parameters of the MXT mouse mammary tumor.

With the use of an in vivo tritiated thymidine ([3H]dThd) nuclear labeling followed by autoradiography, the effects at different times before sacrifice of single or paired injections of 17 beta-estradiol (E2) and/or progesterone (Pg), at different concentrations, were studied in (C57BL x DBA/2f)F1 (B6D2F1) mice transplanted with the MXT hormone-sensitive mammary tumor. Uteri were chosen as controls for the methodology. With regard to MXT tumors, E2 (0.25, 2.50, or 25.00 micrograms/animal) or Pg (125, 600, or 5,000 micrograms/animal) exerts almost a similar mitogenic influence that is dose related to the former and nor for the later, at least within the range of concentrations studied here; and the mitogenic effect of Pg seems to appear earlier than that of E2. When these steroids are concomitantly given, no obvious synergistic or antagonistic effect can be observed. A second E2 administration seems to have a less pronounced effect on cell proliferation as compared to that exerted by the first injection performed 12 or 24 hours earlier. On the contrary, the repetition of a Pg treatment would rather exert an additive or even a synergistic effect on the tumoral dThd labeling indices with regard to the total duration of stimulation. When one of these two steroids is administered first, it blocks totally the mitogenic effect of the second, provided the latter is given 24 hours later. It is concluded that E2 and Pg have almost a similar mitogenic effect on the MXT tumor and that these hormones exert complex interactions that are probably very important for the growth regulation of this cancer. Further investigations are needed to better understand the precise biochemical mechanisms involved in the modulating actions of these steroids on the MXT mammary growth.

Effect of estradiol on proliferation and on cell loss in the MXT mouse mammary tumor.

The MXT tumor is an experimental mammary tumor, maintained by transplantation in B6D2F1 mice, which contains significant amounts of estrogen and progesterone receptors. The aim of the present work was to study the effect of estradiol (E2) administration on the cell kinetic parameters (thymidine labeling index: TLI, S phase duration: TS, cell cycle duration: Tc, growth fraction: GF, potential doubling time: T, cell loss factor: luminal diameter) of an MXT anaplastic carcinoma strain. These parameters were analyzed using single-, double- or repetitive-tritiated thymidine (3H-TdR) labeling techniques. Uteri were used as controls for the methodology. Our data clearly demonstrate that E2 exerted a marked stimulatory effect on the tumor cell proliferation. This effect is the net result of a shortening of the mean cell cycle duration (Tc) and of a 3-fold increase of the number of cells produced per time unit in 89% of the total cancer cell population. Beside this stimulatory effect, E2 produces a marked increase of the tumoral cell loss. This loss seems to affect a cell fraction which has reached the quiescent state (Q) for a short time.

Phenotypic change of the transplantable MXT mammary adenocarcinoma into mixed bone producing sarcoma-like tumors.

The B6D2F1 mouse mammary adenocarcinoma was adapted to grow in vitro as monolayer. After in vitro passaging of tumor cells, phenotypic changes occurred that were expressed in vivo. Following intraperitoneal inoculation of tumor cells, bone-forming tumors developed. These tumors consisted of undifferentiated adenocarcinoma mixed with large amount of cartilagenous and osseous tissue. The etiology of these phenotypic changes was not yet determined. However, hypothesis of the possible origin of the cartilage and bone forming tissue was formulated. The biologic characterization of the intraperitoneally bone-forming tumor was achieved and the experimental conditions to preserve and induce the reproducible sarcoma-like bone forming tumors were defined. Our data support the usefulness of this new original model for fundamental research as well as for screening of anticancer drugs.

Effect of progesterone on cell proliferation in the MXT mouse hormone-sensitive mammary neoplasm.

The transplantable MXT mouse mammary tumor is an experimental model for which the growth is modulated by ovarian hormones; because of scant knowledge about the effects of progesterone (Pg) alone on cancer cells, the present investigation was made. By measuring the percentage of cells with labeled nuclei after tritiated thymidine ([3H]dThd) injection 1 hour prior to sacrifice [dThd labeling index (TLI)], a study was made of the effect (in vivo) of a near physiologic dose of Pg (125 micrograms ip) on cell proliferation in uteri (as the control Pg target organ) and on tumors of spayed (C57BL female X DBA/2F male)F1 mice. Pg induced a significant and transient rise in TLI, which increased from the 12th to the 24th hour after its injection. This mitogenic effect on tumors was comparable to that elicited by 0.25 microgram 17 beta-estradiol injected under similar conditions. In vivo brief exposure of castrated mice to Pg induced a significant mitogenic effect on MXT tumors. These observations might have important consequences for a better understanding of the endocrine mechanisms involved in human hormone-dependent breast cancer.

Effects of aminoglutethimide plus hydrocortisone on the genital tract of postmenopausal women with advanced breast cancer. A clinical and cytologic survey.

In this study we compared the effects of either Aminoglutethimide (AGL) or Tamoxifen (TAM) therapy on the genital tract of postmenopausal women with advanced breast cancer. Thus, 15 patients treated with AGL, and 10 patients treated with TAM underwent gynaecological examination, during which vaginal smears were taken. All smears were reviewed in blind by one pathologist for determination of karyopycnotic indices (KI). Under TAM, no significant clinical abnormality was observed, except in one patient who had a small (histologically benign) endocervical polyp, easily removed during the examination. As reported by others, smears made under TAM therapy were generally characterized by high KI, indicating that an hormonal, estrogen-like stimulation remained present in these patients. On the contrary, most women under AGL had some evidence of vulvovaginal atrophy, which was unvariably associated with low KI on smears. Among the latter, four had severe dystrophic lesions consisting of leukoplasia (1), kraurosis (2) or lichen sclerous and atrophicus (1). It is therefore recommended not to neglect the systematic practice of gynaecological examination in patients with advanced breast cancer under endocrine therapy. These observations also indicate that AGL and TAM exert entirely opposite effects on the vaginal mucosa, which is a very sensitive estrogen-target tissue. In good agreement with former endocrine studies, AGL acts as a potent suppressor of estrogens resulting in severe mucosal atrophy. On the contrary, TAM seems nearly always to display some agonistic hormonal stimulation.

Aromatase, 17 beta-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.

Induction of progesterone receptor in an estrogen, progesterone receptor-negative breast cancer cell line.

In MCF-7 cell culture, some sera endow estradiol-17 beta with strong growth promoting properties ("active" sera) while other fail to display this property ("inactive" sera). Passage from "inactive" to "active" sera are shown here to induce the appearance of a progestin binding capacity in the receptor negative line Evsa-T. Competition with various unlabeled steroids established the specificity of this binding reaction. The induction of progesterone receptor required neither estrogens, nor ER and failed to confer major growth sensitivity to hormonal steroids: only medroxyprogesterone acetate was slightly inhibitory at high concentration. These observations disclose the influence of seric factors independent of estrogens and of ER-related mechanisms on PgR induction.

Morphology of MXT mouse mammary tumors. Correlation with growth characteristics and hormone sensitivity.

The transplantable MXT mouse mammary tumor has been a useful tool for studying endocrine mechanisms underlying mammary tumor growth. It is our experience, however, that this model is unstable during serial transplantation. This paper analyses this variability from the viewpoints of histology and estrogen receptor content and indicates that these parameters should always be checked before planning experimental work. It is advised that a more homogeneous material is needed and that this goal should be achieved by clonal selection before transplantation.

Aminoglutethimide and estrogenic stimulation before chemotherapy for treatment of advanced breast cancer. Preliminary results of a phase II study conducted by the E.O.R.T.C. Breast Cancer Cooperative Group.

While both endocrine therapy and chemotherapy are of proven value in the treatment of advanced breast cancer, the effects of combining these two methods or applying them consecutively have been relatively disappointing. This may be due to endocrine therapy suppressing cell division, in hormone-dependent tumors, whereas chemotherapy acts mainly on active-dividing cells. A trial protocol has therefore been devised which seeks to exploit the properties of both types of therapy. Oestrogen suppression is first obtained by aminoglutethimide (Orimeten) plus hydrocortisone; after 2 weeks, ethinyloestradiol is given to induce cell division and followed 24 h later by a combination of 3 cytotoxic agents given intravenously. This pattern of therapy, repeated at regular intervals, appears to be producing favorable clinical results. A phase-III study is being started among patients with hormone-dependent advanced breast cancer.

In vitro synergism between estrogens and cytotoxic agents.

Two lines of research intending to achieve synergism between cytotoxic agents and estrogens for breast cancer treatment are pursued in our laboratory. According to a screening procedure we select cytotoxic-linked estrogens which bind to estrogen receptors and thereby would be specifically concentrated into the tumor cells. A mesylate derivative of estrone has emerged from our investigations. This compound displays a very strong binding affinity for the receptors and inhibits the growth of the receptor-positive MCF-7 breast cancer cell line. The lack of growth inhibition in the receptor-negative line Evsa-T indicates that it is devoid of major non-specific cytotoxicity. We attempt to enhance the vulnerability of the tumor cells by producing an estrogen-induced modification of their chromatin. The in vitro exposure of isolated uterine nuclei to uterine cytosol preincubated with estradiol increases their ability to bind [3H]actinomycin D. Identical results are obtained with MXT mouse mammary tumors. Experiments are in progress to settle whether these changes are associated with enhanced cell killing.

Assay for estrogen and progesterone receptors of breast cancer cell lines in monolayer culture.

A whole-cell assay for measuring estrogen (ER) and progesterone (PgR) receptors in monolayer culture of human breast cancer cell lines is described. It is based on the measurement of incorporated tritiated ligands during 50 min of incubation (i.e. [3H]estradiol for ER, [3H]ORG-2058 for PgR). The assay fulfills all criteria of specificity as shown by competitive studies and measurements of the dissociation constants of the binding reactions. Moreover, a subcellular fractionation of MCF-7 labeled cells revealed that the majority of incorporated steroids was associated with the nuclear fraction. This finding is consistent with the concept of nuclear location of steroid-receptor complexes. Cultures in the presence of 10(-8) M estradiol indicated that the methodology is adequate for detecting the well-known estrogenic induction of PgR synthesis. The assay proved suitable for the quantitative assessment of the receptor content of various neoplastic (MCF-7; ZR-75-1, Cama-1, Evsa-T) and non-neoplastic (HBL-100) cell lines. The methodology has the other advantages of being simple and rapid, of requiring small amounts of cells and of allowing histological examination of the latter before, during and after biochemical analysis.

Effect of castration and 17 beta-estradiol pulse on cell proliferation in the uterus and the MXT mouse mammary tumor.

The effects of a single 17 beta-estradiol (E2) injection on cell proliferation were studied in 3 groups of 30 mice transplanted with the MXT ovary-dependent mammary tumor. In group A, all animals were castrated prior to tumor implantation; groups B and C had intact ovaries at the time of transplantation, but group B was left intact throughout the experiment, while group C underwent castration 4 weeks later. On day 40 in groups B and C and on day 80 in group A, in which tumor development was significantly delayed, the same procedure for testing the effects of E2 was applied: Ten controls received 0.1 ml saline ip and were killed on the next day; 4 lots of 5 mice received 0.25 micrograms E2 ip and were killed one by one at 12-hour intervals. Exactly 1 hour prior to sacrifice, each animal received 25 microCi [methyl-3H]thymidine ip. Histologic sections of tumors and uteri were processed for autoradiography, and nuclear thymidine (dThd) labeling indices (LI) were determined. All tumors of group A grafted under unfavorable hormonal conditions were poorly differentiated, and E2 injection induced no appreciable changes in their dThd LI. Tumors B and C were well-differentiated adenocarcinomas, in which E2 induced significant modifications of cell proliferation. In group B, complex changes in dThd LI occurred in tumors as well as in uteri, probably due to interferences with the ovarian hormonal production. In group C, E2 produced a marked rise in dThd LI in tumors, lasting from the 12th to the 36th hour after its injection. Stimulation was maximum at the 24th hour, representing a 2.8-fold increase over mean basal dThd LI. It is concluded that the presence of an intact ovarian function at the time of transplantation is critical for maintaining the properties of hormone dependence in MXT tumors. In mice castrated after tumor implantation, a single E2 injection induces a marked and partially synchronous proliferation of neoplastic cells. The hypothesis that such hormonal manipulation might amplify the killing effect of cell cycle- or phase-specific cytotoxic drugs could be adequately tested with this model.

Cisplatin plus vindesine in advanced breast cancer: a phase II trial of the EORTC Breast Cancer Cooperative Group.

This phase II clinical trial was conducted in a series of patients with advanced breast cancer, refractory to conventional chemotherapy. The therapeutic regimen consisted of a combination of cisplatin 100 mg/m2, given as a 24-hr infusion on day 1 and vindesine (VDS) 2 mg/m2, i.v. bolus on days 1 and 8. VDS injection was omitted on day 8 in patients with poor bone marrow reserves (prior extensive irradiation). Courses were repeated at 4-week intervals until documented disease progression. Among 46 evaluable patients, there were two complete and seven partial remissions for an overall response rate of 20%. These responses lasted for a median of 21 weeks (range 8-89 weeks). Remission rates according to the predominant metastatic site were as follows: soft tissue, 3/8 (38%); bone, 0/6 (0%); viscera, 6/32 (19%). Transient myelosuppression and gastrointestinal intolerance were almost universal. Renal function impairment and neurologic manifestations were frequently encountered but these adverse reactions were generally mild. Significant antineoplastic activity in far-advanced and heavily pretreated patients warrants further evaluation of this regimen at an earlier stage of the disease.