A combined histological, immunocytochemical and biochemical approach in the evaluation of estrogen receptors in breast carcinomas

Correlation of structural and functional data might lead to better identification of hormone-dependent tumors. Sixty breast cancer specimens, sent to the biochemistry laboratory for estrogen receptor (ER) analysis, were studied here by a combined morpho-functional approach. Histological examination of needle biopsies on frozen tissue blocks showed that 12 cases (10%) were free of tumor cells; these cases mostly proved ER negative. On the other 48 cases, an immunocytochemical reaction was performed on the biopsy sections with a monoclonal antibody directed against p 29, an estrogen receptor related antigen. The staining values for p 29 and the biochemical ER findings were significantly correlated. A combined histological, immunocytochemical study seems to offer advantages in the selection of patients for hormonal therapy.     

Image cytometry of estrogen receptors in breast carcinomas

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.     

Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies

The subcellular localization of estradiol receptor (ER) has been examined using various experimental approaches. Immunocytochemical studies using the monoclonal antibody JS 34/32, raised against calf uterine cytosolic ER, yielded only equivocal results. In general, cells and tissues pretreated with estradiol showed positive immunostaining in the nuclei whereas those not exposed to the steroid did not show any staining. Nuclear translocation of ER was examined in intact MCF-7 cells using compounds which are known to influence receptor activation. When MCF-7 cells were exposed to molybdate (20 mM), nuclear translocation was completely inhibited while dithiothreitol (20 mM), dibutyryl cAMP (1 microM) and dibutyryl cGMP (1 microM) increased the translocation 2-3-fold. Phenol red, at the range of concentrations generally used in tissue culture media, also increased translocation. The physiological validity of such translocation was examined using cellular progesterone receptor (PR) synthesis as a specific parameter. When MCF-7 cells were grown in media containing phenol red for 48 h, the PR synthesis increased significantly. We further examined whether cytoskeletal proteins are involved in the translocation of ER. Colchicine, an inhibitor of microtubule assembly, inhibited translocation of ER in MCF-7 cells at 1-10 microM. PR synthesis was also inhibited by colchicine in a dose-dependent manner. It may be concluded from these and other published data that ER may not be located at all times in a single subcellular compartment but may rather exist in a dynamic equilibrium between the plasma membrane, cytoplasm and nucleus.     

Correlation of immunocytochemical and immunohistochemical determination of estrogen and progesterone receptors in breast cancer

OBJECTIVE: To evaluate prospectively the degree of correlation between immunocytochemical (ICC) and immunohistochemical (IHC) determination of estrogen (ER) and progesterone receptors (PR) in breast cancer. STUDY DESIGN: Fine needle aspiration cytology (FNAC) of 101 primary breast cancers were immunostained for ER and PR. They were compared with similar determinations in formalin-fixed paraffin sections of biopsies from the same patients. In cases of discrepancy, the histologic result was considered the gold standard. RESULTS: For ER a cytohistologic correlation of 94%, with a sensitivity of 96.1% and specificity of 86.9%, was found. For PR the cytohistologic correlation was 71.2%, with a sensitivity of 65.7% and specificity of 83.8%. CONCLUSION: ICC determination of hormone receptors in routinely fixed smears obtained by FNAC is a simple method that correlates adequately with the results of IHC determinations, especially for ER.     

An immunocytochemical method for assaying oestrogen receptors in breast cancers. A comparison with the steroid binding assay

The presence of oestrogen receptors was studied in 105 human breast carcinomas using monoclonal antibodies (Abbott ER-ICA kit). The oestrogen receptors of neoplastic cells were semiquantitatively measured and correlations were made to receptor values determined by a dextran-coated charcoal (DCC) steroid binding assay and to histological grade. Immunoreactive cells were found in about 2/3 of the tumours. Usually only a fraction of the cells within each tumour were immunoreactive, and the staining intensity varied among different cells. In general, well differentiated tumours had a greater proportion of immunoreactive cells than poorly differentiated ones. In most cases (65/98) a good agreement was found between the ER-ICA method and the DCC assay. However, in 33 cases discrepancies were demonstrated.     

Determination of estrogen receptors in cytologic imprints of breast cancers using immunocytochemical methods.

Immunostaining of estrogen receptors (ERs) was carried out on imprints of 62 breast carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique (the Abbott ER-ICA kit). The results were compared to those obtained by the conventional biochemical analysis of cytosol proteins and to the degree of tumor differentiation. The cytologic specimens were insufficient for analysis in 6 cases; of the remaining 56 cases, 37 (66%) showed a positive ER reaction. In 51 cases with both types of ER analysis, the immunocytochemical staining of the imprints correlated strongly with the biochemical analysis in 44 cases and weakly in 3. Four cases were negative immunocytochemically and positive biochemically. Among the ductal carcinomas, well-differentiated tumors had higher percentages of ER-positive cells than did poorly differentiated tumors. These results show that the immunoperoxidase method is a highly specific and sensitive technique for the evaluation of ER content; it may be applicable to small samples of tumor tissue and may provide additional information for identifying hormonally responsive breast tumors.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

Alterations of estrogen receptors, progesterone receptors and c-erbB2 oncogene protein expression in ductal carcinomas of the breast

Estrogens are important for stimulating the growth of a large proportion of breast cancers. Progesterone plays critical roles in breast development and tumorigenesis. The c-erbB2 gene (HER-2/neu) is a proto-oncogene expressed in 10-34% of breast cancers. Its expression is associated with poor clinical outcome. The hypothesis that the progression of in situ ductal carcinoma of breast to invasive ductal carcinoma is associated with alterations of ER, PgR and HER-2/neu protein expression was tested. Of 100 mastectomy specimens examined, all contained both ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC) not otherwise specified (NOS). The status of ER, PgR and HER-2/neu proteins was examined by immunochemistry. ER and PgR protein expression was scored as the mean value of positively stained cells. HER-2/neu protein expression was evaluated on ts staining pattern (0, 1+, 2+ and 3+). We found variations between DCIS and IDC with significant decrease of the mean values of ER and PgR positively stained cells in high-grade (Grade 3) IDC (ER: 49.2+/-10.3 vs. 30.8+/-5.5 and PgR: 40.0+/-10.0 vs. 22.3+/-5.1 in DCIS and IDC, respectively, P<0.05). Invasive carcinomas with lymph node metastases or lymphovascular invasion or both had lower mean values of ER and PgR positively stained cells compared to those without these features. In IDC (Grade 3), HER-2/neu protein expression values (1.2+/-0.2) were significantly high compared to DCIS (0.7+/-0.3, P<0.05). In addition, HER-2/neu protein expression values were significantly higher (P<0.05) in IDC with lymph node metastases or lymphovascular invasion (1.5+/-0.3) than those without these features (0.8+/-0.2). A significantly high mean (P<0.05) of ER and PgR positively stained cells was observed in postmenopausal females compared to premenopausal women. In contrast, high HER-2/neu expression values were seen only in premenopausal females. A significant positive correlation was observed between ER and PgR receptor expression (r=0.81). A low degree inverse correlation (r=-0.24, P<0.012) was found between ER+/PgR+ tumors and HER-2/neu expression. These findings substantiate the notion that breast cancer progression is often associated with alterations of ER, PgR and HER-2/neu expression. The underlying mechanisms of these alterations are open for further investigation.     

4-Hydroxytamoxifen-induced cytotoxicity and bisphenol a: Competition for estrogen receptors in human breast cancer cell lines

Summary Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess potentially estrogenic effects of test compounds. As a result, in vitro screening methods such as cell proliferation assays using the estrogen-responsive human breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xenoestrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-induced cytotoxicity. This method measures the ability of various estrogenic compounds to compete with a known estrogen-receptor-mediated antihormonal drug, 4-hydroxytamoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamoxifen treatment of cells results in a dramatic decrease in mitochondrial dehydrogenase activity which is directly related to their estrogen-receptor content, inhibition of this effect with estrogenic compounds represents an estrogen-receptor interaction, or estrogenic rescue. The estrogenic compounds tested include a weak xenoestrogen, bisphernol A (BPA), and two biological estrogens, 17α- and 17β-estradiol. Competitive inhibition of 4-hydroxytamoxifen-induced cytotoxicity by BPA was compared to that of the biological estrogens. The results indicate that the biological estrogens can successfully compete with the antiestrogen in a dose-dependent manner. In addition, the assay is sensitive enough to detect estrogenic rescue by even the very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simple in vitro method could be used as an alternative or second-line screen for potential xenoestrogens.     

New method for the determination of estrogen and progesterone receptors in human breast cell lines

A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, [3H]-17 beta-Estradiol or [3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

Comparison of estrogens and estrogen metabolites in human breast tissue and urine

Background  

An important aspect of the link between estrogen and breast cancer is whether urinary estrogen levels are representative of the intra-tissue levels of bioavailable estrogens.     

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.     

Comparison of estrogen receptor immunocytochemical assay in frozen and paraffin sections.

Frozen and paraffin sections of 11 breast carcinomas were stained for estrogen receptors (ER) using the same rat monoclonal primary antibody, D75P3, as the marker and alkaline phosphatase as the chromogen-linking enzyme. The results of this staining process were assessed visually and with the microTICAS image analysis system to determine the degree of correlation between frozen and paraffin-embedded tissue. In all specimens, some fraction of the nuclei stained positively. This included two specimens selected for their biochemically negative assay; one of them stained strongly positively with D75P3. The results of quantitative analysis support the visually apparent correlation between the two types of samples in terms of both overall staining pattern and intensity of nuclear staining. Although the conclusions of this pilot study are limited because of the small number of cases, this method of staining establishes the feasibility of representative ER determination in archival paraffin-processed material. The additional information provided by this method is potentially useful in stratifying patients in prospective studies on the basis of the efficacy of hormonal therapy in biochemically ER positive breast tumors.     

Immunochemical analyses of estrogen receptors in human breast tumors by a novel monoclonal estrogen receptor antibody (EVG F9)

The objective of this study was to assess the potential utility of a new site-directed, monoclonal anti-estrogen receptor antibody (EVG F9) in detection and analyses of human breast tumor estrogen receptor (ERalpha), using immunoblotting and immunohistochemical assays. Using Western Blot analyses, we demonstrated that EVG F9 monoclonal antibody binds specifically to ERalpha and does not cross-react with ERbeta. Furthermore, binding of EVG F9 to ERalpha was effectively displaced with the immunogenic peptide in Western Blots and in immunohistochemical analyses. In Western Blot analyses, EVG F9 detected ERalpha at low concentrations approaching 5 to 10 fmol/sample. Determination of ERalpha status of a series of human breast tumor samples by Western Blot analyses or immunohistochemistry using EVG F9 correlated well with ERalpha values measured by ligand binding assays. These observations suggest that EVG F9 monoclonal anti-ERalpha antibody is a valuable immunochemical tool for detection and analyses of ERalpha in human breast tumors.     

The influence of enzymes on the estrogen receptors of human uterus and breast carcinoma

The influence of hydrolytic enzymes on the estrogen receptor and on the estradiol-receptor complex was studied. Proteolytic enzymes (trypsin, α-chymotrypsin, papain) phospholipases A and C, and β-glucuronidase reduced the subsequent in vitro binding of estradiol-17β by the receptor; they also released bound estradiol-17β from the estradiol-receptor complex. In addition, some glycosidases had an effect on the free receptor only. The results indicate that the estrogen receptor may contain phosphorus and carbohydrate moieties.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.