Estradiol receptor and prognostic parameters of human breast cancer.

Estradiol receptors are regarded to predict a likely success of hormonal therapeutic efforts and the prognosis of breast cancer patients. But today its prognostic importance is controversial, discussed as either reflecting intrinsic property of the tumor tissue or better therapeutic accessibility of receptor positive tumors. Moreover, the most important clinical prognosticators--tumor size and axillary lymph node involvement do not seem to be related to the estradiol receptor status. In our investigation, the length of disease free interval is similar in estradiol receptor positive and negative patients and in all sites of distant metastases, but it is significantly reduced if more than 4 axillary lymph nodes are involved. Post recurrence survival is significantly longer in estradiol receptor positive than negative patients and also in patients treated by tamoxifen containing therapies. Its length is independent of the number of axillary lymph node metastases and the type of distant metastases, with a tendency to be longer in estradiol receptor positive than negative patients. In addition, the overall survival is longer for estradiol receptor positive than negative patients and becomes reduced with more than 4 axillary lymph node metastases. Frequency of deaths in estradiol receptor positive patients is half that of negative subjects. Furthermore, the length of overall survival is independent on the type of distant metastases, with tendency to be longer in estradiol receptor positive than negative patients. Longest overall survival could be observed for estradiol receptor positive patients who got therapy regimens containing tamoxifen. The weak prognostic advantages of estradiol receptor positive patients are interpreted by estradiol receptors as intrinsic parameters of breast cancer tissue characterizing more its biological behavior than therapeutic accessibility.

Steroid hormone receptors and antineoplastic chemotherapy in human breast cancer.

Several clinical and experimental investigations suggest that the action of antineoplastic chemotherapy in premenopausal women influences the menopause. Such hormonal reactions are mediated via specific steroid hormone receptors. Therefore, connections between hormone receptors and antineoplastic chemotherapy can be assumed making possible to predict success of chemotherapy on the basis of receptor status. Nevertheless, clinical experiences and animal and cell culture experiments yielded controversial results. This was related to the predictive value of receptor status as well as to the benefits of combined hormone and chemotherapy treatments in concurrent or sequential form. It is undeniable that a displacing of steroidal ligand from its receptors by the usual antineoplastic drugs does not occur. Furthermore, the receptor levels remain unchanged after a treatment with antineoplastic drugs. Thus, the mechanism of action of chemotherapeutic drugs is not related directly to the presence or absence of steroid hormone receptors. Despite this fact the receptor status in chemotherapeutic regimes seems to be helpful to define low or high risk patients. Influences on the ER de-novo-synthesis, actions related to parameters representing reduced tumor growth rates, down-regulation of the receptor gene expression or via receptor mediated hormonal actions to other genes, like the apoptosis-related gene bcl-2, are thought to be possible mechanisms of action of antineoplastic drugs on steroid hormone receptors. Future investigations should monitor the ratios between exon lacking receptor variants and the wild-type receptor during chemotherapy or the control of a ligand uptake during chemotherapy by means of the positron emission tomography.

Steroid hormone receptors related to parameters characterizing the biology of human breast cancer.

Steroid hormone receptors are important parameters to characterize breast tumors. Thus, it is important to evaluate their relationships with factors such as histological type and size of the tumor, axillary lymph node invasion and distant spread, age and menopausal status of the patients, and parameters of tumor differentiation. The receptor levels observed vary within wide ranges. Therefore, statistically significant differences between different groups of parameters are seldom found. A significant dependence on receptor levels has been observed only for patient age and menopausal status. The parameters clinical stage, tumor size, tumor histology, metastatic involvement and histopathologic grading showed no statistically significant relation with receptor levels. Nevertheless, a relationship exists between all parameters mentioned and the frequency of a positive receptor status. Consequently, receptor status can contribute to define the biological behavior of the disease in specific groups of patients. In individual cases other parameters than the biochemical evidence of steroid receptor binding seem to be more important. We found that data derived from DNA flow cytometric measurements allowed a better recognition of tumor aggressiveness than ER status. In axillary lymph node metastases we usually observed higher receptor levels than in primary tumors, but we did not find an age dependency of ER levels in these metastases.

Estradiol receptors and metastasis in human breast cancer.

The localization and extent of metastasis determined the prognosis of breast cancer in a decisive manner. Thereby axillary lymph node involvement represents one of the most important prognostic indicators. The estradiol receptor status is also attributed some prognostic importance. There might therefore be relations between these prognostic factors. However, the majority of investigators could not find a correlation between the extent and timing of regional lymph node involvement and estradiol receptor status. In contrast, there are numerous findings which confirm correlations between estradiol receptors and the localization, extent, and timing of distant metastasis. The findings obtained in more recent years have been collected and discussed in relation to events included in the process of metastasis such as release of proteases and existence of receptor variants.

Cytokinetic investigations in human breast cancer by flow cytometrically recorded DNA/protein distributions.

This prospective study characterizes T1-T2 breast carcinomas (N = 114) and fibroadenomas (N = 16) by cell kinetic parameters derived from flow cytometrically recorded DNA/protein histograms. Ploidy level, cell cycle distribution and the number of cell subpopulations (SP) characterized by correlating DNA and protein values were assessed. The subpopulations were derived from the three-dimensional plot. The estrogen receptor (ER) status was determined biochemically (N = 61). Within the G1/0 cell peak 1-6 SP were evident in principle. Depending on the number of SP, two subsets were established: subset 1 with < or = 2 SP, subset 2 with > or = 3 SP. They differed significantly in proliferative activity expressed in the percentage of cells in the G2M phase. Subset 2 showed the higher activity. Analysis of subset distributions revealed that subset 1 prevails in favourable prognostic cases as ER positive cases (P < 0.03), lobular carcinomas (P < 0.01) and LN- cases (P < 0.03), whereas subset 2 prevails in the unfavourable counterparts. Analysis of variance showed that the main effect on proliferative activity indicated by G2M% is due to subpopulation composition rather than histologic type, nodal status or ER status (P < 0.01, P < 0.002, P < 0.05), not even due to ploidy level (P < 0.0001). The rationale for subset stratification may be cytogenetic variability connected with protein content heterogeneity accounting for kinetic SP.

Steroid hormone levels in patients with advanced breast cancer during therapy with droloxifene: a pilot study.

Antiestrogens, particularly tamoxifen, are effective in the treatment of pre- and postmenopausal women suffering from all stages of breast cancer. Unfortunately, many patients become resistant to tamoxifen during therapy, which allows the tumor to progress. Thus, a preclinical recognition of tumor progression, i.e. by monitoring serum hormone levels, could be worthwhile. The serum levels of dehydroepiandrosterone sulfate and estradiol of postmenopausal women with advanced breast cancer treated by the new antiestrogen droloxifene were therefore checked. However, only non-significant changes in the hormone levels during droloxifene therapy were observed, and no relation was found between hormone levels and the course of the disease, success or exhaustion of droloxifene application, or development of tumor progression. Our data do not confirm earlier findings reported in the literature that measurement of hormones seems to be suitable for an early indication of tumor progression during an antiestrogen therapy before its clinical manifestation.

Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident. The C. albicans isolates from the HIV-positive group that were characterized by higher levels of proteinase activity were also less susceptible to the widely used azole antifungal ketoconazole and fluconazole. Collectively, the present data are consistent with a concept of early preferential selection of a subpopulation of C. albicans in HIV-infected patients.

[Tamoxifen flare]. / Tamoxifen-flare.

Tamoxifen flare is a relatively seldom occurring phenomenon but it can force to therapeutical consequences. In every case a careful control of patients has to perform. As yet do not exist clear definitions as well as precise explanations of mechanism of action of flare and tamoxifen withdrawal response. These are related to normal metabolism of tamoxifen and can not explain satisfying flare occurrence in a small number of patients only. By reason of clinical experiences in most cases flare can be regard a sign for hormone sensitivity of the tumor and its following regression. But again do not exist clear explanations for this fact.

Estradiol receptor and prognosis in human breast cancer and its metastases.

Follow-up of 86 patients for at least 40 months confirmed age dependency of receptor binding capacity in primary breast tumors but not in lymph node metastases. In most cases receptor binding capacity was higher in lymph node metastases than in primary tumors. Prognosis of the disease expressed as percentage of patients who survived and had a disease-free interval is related to receptor binding capacity of the primary tumors investigated. In our investigation, a value of 60 fmol/mg protein seems to be a suitable cut-off value to distinguish between breast cancer patients with good and bad prognosis. Receptor levels of lymph node metastases showed a similar behaviour as those of primary tumors related to percentage of patients who survived, but not to disease-free interval.

Enrichment of estradiol receptor from calf uterus.

A three-step procedure to enrich estradiol receptor protein from calf uterus nearly 30,000-fold has been described. Over-all yield is 12%. Control of the single steps has been performed by sodium dodecyl sulfate-electrophoresis, sucrose density gradient centrifugation, gel filtration and determination of receptor quantity. Immunological properties of the preparation obtained have not been controlled.

Chromatographic investigations of estradiol receptor from breast cancer tissue.

DEAE-cellulose chromatography of non-activated and activated estradiol receptors yields a different, characteristic elution pattern. This chromatography should therefore be able to demonstrate the presence of intact receptors. For clinical receptor investigations, it is usual to select arbitrary cut-off values for receptor quantity and Kd to classify into receptor positive and negative cases. DEAE-cellulose chromatography confirmed the results obtained by such classification in most cases, except those which had been classified as negative on the basis of Kd values higher than 2 nM. In these cases, DEAE-cellulose chromatography indicated the presence of estradiol receptors and therefore a misclassification of the measured results. Thus DEAE-cellulose chromatography can contribute to a clarification of the equivocal results. On the other hand, we did not get any indications of damages to the receptor molecule which in our previous investigations were found to be the result of violent homogenization procedures.

Investigations of droloxifene and other hormone manipulations on N-nitrosomethylurea-induced rat mammary tumours. 1. Influence on tumour growth.

The effect of droloxifene, a new anti-oestrogenic drug, on N-nitrosomethylurea-induced mammary tumours of Sprague-Dawley rats was investigated and compared with that of tamoxifen. The response of tumour growth to ovariectomy or to treatment with aminoglutethimide or high doses of oestradiol was also studied. Ovariectomy was by far the most effective treatment for mammary-tumour-bearing animals. More than 75% of the tumours in ovariectomized rats did not grow progressively but remained in remission for up to 12 weeks after castration when the experiment was terminated. The inhibitory effects of droloxifene and tamoxifen on mammary tumour growth were similar, but body weight loss of animals treated with tamoxifen was more marked than that of animals treated with droloxifene at the same dose and schedule.

Investigations of droloxifene and other hormonal manipulations on N-nitrosomethylurea-induced rat mammary tumours. 2. Influence on oestrogen receptor.

In N-nitrosomethylurea-induced rat mammary tumours, tamoxifen is found to compete at the binding sites of the oestradiol receptor if a receptor determination is performed 1 day following the last drug application to animals. Despite a higher binding affinity of droloxifene (3-OH-tamoxifen) to oestradiol receptor, compared to tamoxifen, its influence on the measurable receptor quantity is only very weak or not demonstrable. Therefore, binding affinity is not a valid explanation for the different influences of the two anti-oestrogens on the receptor. These only can be attributed to different behaviour patterns of both substances in relation to their half-lives and metabolism and accumulation in the organism. Owing to the short half-life of droloxifene, even 1 day after the last application too little drug is available to compete for oestradiol binding sites. In the case of both anti-oestrogenic substances, cessation of drug application for 8 weeks abolished any influence on the oestradiol receptor. Furthermore, failure of aminoglutethimide to influence the oestradiol receptor could be observed because this substance does not act via this receptor. The experiments performed confirm literature data regarding the effect of aminoglutethimide therapy on oestradiol receptors in breast tumour tissue of human beings. In summary: receptor investigations of N-nitrosomethylurea-induced rat mammary tumours, used as a model to test therapy regimens with droloxifene or other drugs with a short half-life, may be of limited value only.

Characterization of two human mammary carcinomas, MT-1 and MT-3, suitable for in vivo testing of ether lipids and their derivatives.

The human mammary carcinomas MT-1 and MT-3 originate from surgical material and were transplanted in nude mice. Both tumors have been classified as estradiol- and progesterone receptor-negative. Therapeutic doses of hormones and anti-hormones remained without growth inhibitory effect. MT-1 and MT-3 proved to be sensitive to conventional cytostatic drugs used for treatment of mammary carcinomas; striking is their sensitivity to ether lipids. Therefore, they are considered suitable tumor models for this class of substances.

[Multicenter pilot studies on the determination of steroid hormone receptors]. / Multizentrische Pilotstudien zur Bestimmung von Steroidhormonrezeptoren.

Corresponding the international tendency two trials have been performed in GDR to compare results of hormone receptor determinations with prepared probes by several laboratories. As probes we used lyophilized calf uterus. Methods applied for receptor determinations based on dextran charcoal technique, but allowed modalities developed in the various laboratories. In consequence of extensive scattering of results got during the first trial an additional investigation basing on a standard procedure was performed in the second trial. Using this standard procedure better corresponding results could be found in comparison to investigations following the laboratory modalities. Consequently application of a standardized technique in all laboratories should be demanded. Final aim of these efforts is to obtain a reliable semiquantitative classification of low, medium and high receptor contents in the tissue probes investigated.

Activation of the estrogen receptor from N-nitrosomethylurea-induced rat mammary tumors.

The activation of the estrogen receptor (ER) from N-nitrosomethylurea (NMU)-induced rat mammary tumors was studied in vitro. The activation of the receptor induced by heating of the cytosol containing occupied ER was measured by a 3-4-fold increase of receptor binding to nuclei in comparison with the nuclear binding of the nonactivated ER. The activation of the ER was further shown by alteration of the elution profile from DEAE-cellulose. A shift of the receptor peak from 234 mM (Peak II, nonactivated ER) to 70 mM (Peak I, activated ER) phosphate buffer could be obtained. The overall recoveries of activated ER following chromatography on DEAE-cellulose were significantly lower than the recoveries of the nonactivated ER, 71 and 85%, respectively. Binding of the activated ER to nuclei and chromatography of the supernatant which is not able to bind to nuclei on DEAE-cellulose resulted in a decrease of Peak I and in an increase of the overall recovery. These findings suggest that the nuclear bound ER consists of two parts. One is represented partially by Peak I of the elution profile and the other one by that part of the receptor which can not be eluted from the column under the conditions used. Furthermore, the dissociation of tritiated estradiol (E3H) from the nonactivated ER followed a two component exponential function whereas after activation a monophasic dissociation curve could be observed. The mean half times for the dissociation of E3H from the activated and nonactivated ER were 101 and 7.2 min, respectively. Finally, the nonactivated molybdate stabilized ER sedimented in 5-20% sucrose density gradients as two peaks, one at 9.5 S and the other at 4 S. After activation of the ER only the smaller 4 S peak was evident. Molybdate inhibited the activation of the ER measured by nuclear binding assays, sucrose density gradient analysis, dissociation kinetics or ion exchange chromatography but not completely in every case.

[Hormonal aspects of benign diseases of the breast]. / Hormonelle Aspekte der benignen Erkrankungen der Mamma.

Basing on references from literature it has been looked for possibilities to characterize benign lesions of the breast by endocrine data and to find a discrimination between types connected with high or low risk for breast cancer. Conventional investigations of hormonal excretions and measurements of plasma contents only provide data for statistical evaluations which can not be used to assess the situation of an individual patient. Obviously one has to look for the processes of hormonal promotion on a cellular level. These processes are thought to realize a non-hormonal initial event to a benign or malignant disease. This requires finding of corresponding representative data. Some possibilities as receptor investigations or recording of hormone induced secretion proteins have been discussed.

Binding of estrogen receptor from N-nitrosomethylurea-induced rat mammary tumors to nuclei.

The binding of the cytoplasmic estrogen receptor (ERc) from N-nitrosomethylurea (NMU)-induced rat mammary tumors to the nucleus using a cell-free system is described. All tumors studied were estrogen-receptor-positive and most of them were hormone-dependent. Sixty-two percent of all tumors investigated (n = 134) decreased in size more than 30% 4-5 days after ovariectomy. Brief heating of the cytosol loaded with tritiated estradiol induced activation of the ERc measured by an increase of nuclear binding activity. Temperature-dependent activation was evident in every case. The optimal time and temperature of activation were 15-60 min at 30 degrees C. After denaturation of the ERc by heating for 20 min at 56 degrees C only small parts of free estradiol could be bound to nuclei. Mg2+ ions and EDTA inhibited the nuclear binding of the receptor. The nuclear binding assay was performed for 1 hr at 0-4 degrees C. After this time the activated ERc was bound nearly maximally to nuclei. Under optimized conditions 50-60% of the ERc could be bound to nuclei maximally. Using the same medium for the preparation of crude and purified nuclei the binding of the receptor to both kinds of nuclei was similar. Na2MoO4 prevented the activation of the ERc from NMU-tumors completely but did not influence the binding of the previously activated receptor to nuclei.

[Correlation of the results of in vitro tests (cell cultures and receptor analyses) of human breast cancers in relation to their hormone dependence]. / Korrelation der Ergebnisse von In-vitro-Tests (Zellkulturen und Rezeptoranalyse) menschlicher Mammakarzinome hinsichtlich ihrer Hormonabhängigkeit.

Pretherapeutic parameters are demanded for the hormonal treatment of metastasing breast cancer. Many methods have proved to be ineffective in the assessment of the hormone sensitivity until now (determination of concentrations of steroid hormones in serum and urine or Barr's sex-chromatin). Investigations carried out hitherto on estradiol receptor content or on hormone sensitivity based on in-vitro cultures were promising. They caused us to compare both methods at the same mamma carcinoma tissue. The results of this study showed that no unequivocal correlation between receptor status and the results of in-vitro cell culture tests for hormone sensitivity could be found. Although all in all the results of both methods are almost identical in percentage, there are however substantial deviations between the results in the directly corresponding comparison of each single tissue sample.