p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant 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.     

Cytologic diagnosis of estrogen and progesterone receptors in breast imprints

OBJECTIVE: To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. STUDY DESIGN: Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. RESULTS: Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. CONCLUSION: ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.     

Nuclear morphometry and estrogen receptors in infiltrating ductal carcinoma of the breast

In this study ten cases of breast infiltrating ductal carcinoma have been considered. In all of them the content of ER has been evaluated by using monoclonal antibodies. Five of them were ER positive and five were ER negative. For the morphometric study ten nuclei of each case have been considered. By using the S.A.M. (Shape Analytical Morphometry) work-station an analytical study of the nuclear shape was performed. The first step was the extraction of fundamental shape which describes the basic shape of original contour without its irregularities. It was obtained by using two parametric equations. The second step was the evaluation of shape asymmetry by S.A.E. (Shape Asymmetry Evaluator). Finally the contour irregularities were evaluated by Fourier analysis. Along with analytical parameters, dimensions (area, perimeter and maximum diameter) were considered too. All obtained data were submitted to univariate statistical analysis (Student's T test) to compare the two groups (ER positive and ER negative tumors). Area, perimeter and maximum diameter were significatively greater in ER negative cases while analytical parameters were not discriminant between the two groups.     

Tumor growth fraction, expression of estrogen and progesterone receptors, p53, bcl-2 and cathepsin D activity in primary ductal invasive breast carcinoma and their axillary lymph node metastases

The aim of this paper is to determine similarities and differences between tumor cell subclones in cases of ductal invasive breast carcinoma, and which occupy primary tumor and local axillary lymph metastases. The tumor growth fraction evaluated by Ki-67 was analyzed along with the expression level of estrogen and progesterone receptors, protein p53, proto-oncogene protein bcl-2 and cathepsin D in 60 patients. Metastatic lymph node in axilla has a higher growth fraction of the tumor cells than the primary tumor (p = 0.045), as well as the higher level of bcl-2 overexpression (p = 0.014). No statistically significant difference was found in the presence of immunohistochemically identified estrogen receptors (p = 0.161) and progesterone receptors (p = 0.081) between the primary tumor and the metastatic lymph node in axilla. Likewise, no difference was found between the immunohistochemical evaluation of p53 (p = 0.356) and cathepsin D activity (p = 0.928). A higher growth fraction of the tumor cells and the higher level of bcl-2 overexpression in metastatic tumor cells indicate the more aggressive cell subclones. This study does not support the routine testing of both primary tumor and locoregional metastasis to evaluate the breast cancer hormone receptor status.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.     

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.     

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.     

乳腺浸润性导管癌组织中CAS蛋白表达的临床病理意义

目的研究乳腺浸润性导管癌组织中细胞凋亡易感蛋白（CAS）表达的临床病理意义。方法选取乳腺浸润性导管癌53例、普通导管增生20例、异型导管增生20例、导管原位癌10例、正常乳腺组织14例,应用免疫组化方法观察CAS蛋白的表达,并探讨CAS与乳腺癌临床病理因素的关系,分析CAS和HER2、ER、PR以及ki-67指数的关系。结果 CAS在正常乳腺、普通导管增生、异型导管增生、导管原位癌、浸润性导管癌中的阳性率逐渐升高,分别为14.3%、25.0%、40.0%、60%、75.5%（P=0.000）,CAS、HER2均与乳腺癌组织学分级、核分裂像、淋巴结转移有关;CAS评分与ki-67指数（r=0.439,P=0.003）和HER2评分（r=0.598,P=0.000）正相关。结论 CAS与乳腺癌的发生、发展、增殖、淋巴结转移有关,可能作为反映乳腺癌生物学行为的肿瘤标记物,CAS蛋白的表达和HER2有一定的相关性。     

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.     

Effects of low-dose tamoxifen on breast cancer biomarkers Ki-67, estrogen and progesterone receptors

Breast carcinoma is the most common malignancy among women and it has a major impact on mortality. Studies of primary chemoprevention with tamoxifen have generated high expectations and considerable success rates. The efficacy of lower doses of tamoxifen is similar to that seen with a standard dose of the drug, and there has been a reduction in healthcare costs and side effects.The immune reaction to monoclonal antibody Ki-67 (MIB-1) and the expression of estrogen receptors (1D5) and progesterone receptors (PgR 636) in breast carcinoma were studied in patients treated with 10 mg of tamoxifen for a period of 14 days.A prospective randomized clinical trial was conducted with 38 patients divided into two groups: Group A: N = 20 (control group-without medication) and Group B: N = 18 (tamoxifen/10 mg/day for 14 days). All patients signed an informed consent term previously approved by both institutions. Patients underwent incisional biopsy before treatment and 14 days later a tumor tissue sample was obtained during surgical treatment. Positivity was quantitatively assessed, counting at least 1.000 cells per slide. For statistical data analysis, a Wilcoxon non-parametric test was used, and α was set at 5%.Both groups (A and B) were considered homogeneous regarding control variables. In Group A (control), there was no statistically significant reduction in Ki-67 (MIB-1) (p = 0.627), estrogen receptor (1D5) (p = 0.296) and progesterone receptor positivity (PgR 636) (p = 0.381).In Group B (tamoxifen 10 mg/day), the mean percentage of nuclei stained by Ki-67 (MIB-1) was 24.69% before and 10.43% after tamoxifen treatment. Mean percentage of nuclei stained by estrogen receptor (1D5) was 59.53% before and 25.99% after tamoxifen treatment. Mean percentage of nuclei stained by progesterone receptor (PgR 636), was 59.34 before and 29.59% after tamoxifen treatment. A statistically significant reduction was found with the three markers (p < 0.001).Tamoxifen significantly reduced monoclonal antibody Ki-67 (MIB-1), estrogen receptor (1D5) and progesterone receptor positivity (PgR 636) in the breast epithelium of carcinoma patients treated with a 10 mg dose of tamoxifen for 14 days.     

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.     

Detection by immunohistochemistry of c-erbB2 oncoprotein in breast carcinomas and benign mammary lesions.

The presence of the c-erbB2 oncoprotein was demonstrated by immunohistochemistry in a study involving 173 mammary lesions. The lesions included infiltrating cancers, non-invasive neoplasia, as well as atypical and benign lesions. Our aim was to investigate the correlation between the c-erbB2 oncoprotein overexpression and the morphological features of the different mammary tissues analyzed to obtain a better characterization for the growth potential of certain lesions, with emphasis placed on the non-invasive neoplasia and the atypical lesions. Nearly 30% of infiltrating ductal carcinomas (27/89 cases) and 2 out of 24 infiltrating lobular carcinomas were positive. The comedocarcinomas were mostly stained (83%). In contrast, the intraductal carcinomas of cribriform or papillar patterns were consistently negative. No staining was observed in the atypical epithelial hyperplasia located in the vicinity of positive cancers for anti-oncoprotein c-erbB2 antibody. Furthermore, the only 5 positive cases for c-erbB2 out of 32 cancer-free cases were three fibroadenomas and two fibrocystic diseases with atypical ductal hyperplasia. A close correlation was thus observed between c-erbB2 oncoprotein overexpression and cancerous cell morphology, characterized by a marked nuclear hypertrophy often associated with cellular pleomorphism. However, predictive abnormalities of malignant transformation in non-neoplastic epithelial proliferation was difficult to identify, considering only the c-erbB2 expression. A group of tumors with little nuclear abnormalities were found positive for c-erbB2 immunostaining. These probably corresponded to a particular cellular phenotype. Further studies involving other oncogenes should lead to a better characterization of the different tumor phenotypes and help to clarify breast carcinogenesis.     

Spatial and molecular aspects of estrogen and progesterone receptor expression in human uteri and uterine carcinomas

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.     

WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors

WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.     

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen. A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor-associated antigen, H23, was compared to expression of genes coding for pS2, c-erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non-breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c-erbB2, ER and pS2, respectively. Non-malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.