Relationship between ER-ICA and conventional steroid receptor assays in human breast cancer

We applied a new immunocytochemical assay for estrogen receptors (ER-ICA) to 82 human breast tumors. Results were correlated with cytosolic estrogen receptors (ERc) and nuclear ER (ERn) determined on the same sample respectively by the radioligand binding assay and by an ER enzyme immunoassay (ER-EIA). All ER-ICA-positive tumors contained more than 10 fmol/mg of protein of ERc and were therefore considered as ERc positive. In contrast, 15.4% of ERc-positive cases were ER-ICA negative. Comparison of ER-ICA results with ERn showed extensive agreement of negativity (92%), whereas 38% of ER-ICA-positive tumors were ER-EIA negative. However, the latter had ERc levels above the positivity threshold. Quantitative features of the immunocytochemical staining such as intensity and percentage of labelled cells, considered separately, did not reflect the amount of ERc or ERn. Cellularity was not significantly correlated with ER-ICA and biochemical results.     

Histopathologic characterization of human breast cancer in correlation with estrogen receptor status. A comparison of immunocytochemical and biochemical analysis

A detailed histopathologic analysis of 399 primary breast carcinomas was performed, and several morphologic features were correlated with the estrogen receptor (ER) status. In all cases ER status was determined immunocytochemically by estrogen receptor immunocytochemical assay (ER-ICA). In 359 carcinomas ER status was also biochemically determined. Invasive lobular, mucoid, and tubular carcinomas rather than ductal carcinomas were ER-positive more frequently in ER-ICA. Medullary and papillary carcinomas had corresponding lower or higher ER positivity, respectively, by both methods. The correlation of histologic grade and its single factors with ER status was statistically significant by both methods. Lymphocytic reaction to tumor showed a significant inverse relationship to ER status by both methods. A statistically significant higher number of ER-positive carcinomas in ER-ICA and dextran-coated charcoal assay (DCC) occurred when elastic tissue was present. Different associations were found between stromal content, tumor diameter, and ER status in DCC and ER-ICA, respectively.     

Immunohistochemical demonstration of estrogen receptors on routine paraffin sections of breast carcinomas: a comparison with frozen sections and an enzyme immunoassay.

In this study the monoclonal antibody ER-ICA (HSpy222) to human estrogen receptor (ER) protein and the peroxidase-antiperoxidase method was used to detect the presence of ER in 83 cryostat sections and in 68 paraffin sections pretreated with pronase in a total of 86 primary breast cancers. In 72 out of the 86 studied cases, a comparative evaluation was performed between the semiquantitative ER-ICA method and the quantitative enzyme immunoassay ER-EIA. A good correlation was found between the semiquantitative ER-ICA results in cryostat and paraffin sections (95.38%; p less than 0.01) in a total of 65 compared cases, concerning both the percentage of ER-positive or negative cells and the staining intensity. In addition, the overall appraisal of the lesion as ER-ICA-positive or ER-ICA-negative as well as the ER-ICA staining intensity and the proportion of ER-ICA stained cancer cells, in both cryostat and paraffin sections, correlated significantly with the mean values of fmol ER/mg determined by the enzyme immunoassay ER-EIA. The performance of the ER-ICA method on paraffin sections as used in the present study proved to be a reliable and reproducible immunohistochemical technique.     

Tumor-proliferative activity, progesterone receptor status, estrogen receptor level, and clinical outcome of estrogen receptor-positive advanced breast cancer

The relations among pretreatment tumor-proliferative activity, progesterone receptor (PgR) status, estrogen receptor (ER) level, and clinical outcome were analyzed in a series of 45 ER-positive advanced breast cancer postmenopausal women treated with tamoxifen (20 mg/day) until disease progression. Tritiated thymidine ([3H]dThd) Labeling Index (LI) by autoradiographic assay was utilized for proliferative activity analysis, whereas the dextran-coated charcoal method was used for ER and PgR evaluation (cutoff value, 10 fmol/mg of protein cytosol). The median [3H]dThd LI value was 1.8%; 73% of cases were PgR positive, and 53% were highly ER positive (greater than 100 fmol/mg of protein cytosol). Clinical responses were more frequently observed in slowly than in quickly proliferating tumors (86% versus 60%; P less than 0.05) but were similar for PgR-positive and -negative cases, as well as for those with high and low ER positivity. Only [3H]dThd LI was found to individualize patients with different survival rates (at 40 mo of follow-up, 78% versus 40% in slowly and quickly proliferating tumors, respectively). The [3H]dThd LI, monitored in ten patients by a second tumor biopsy after 14 days of tamoxifen therapy, was found to have a significantly lower median value (P = 0.03). These data indicate that pretreatment [3H]dThd LI provides information, which is not available in a study of PgR and ER status, on the clinical outcome of ER-positive advanced breast cancer patients treated by hormone therapy.     

Influence of hormones on proliferation of ER-positive cells and ER-negative cells of human breast cancer (MCF-7)

The influence of endocrine therapy on the proliferation of estrogen receptor (ER)-positive cells and ER-negative cells of human breast cancer (MFC-7) serially transplanted into nude mice was analyzed by tumor growth, dextran-coated charcoal (DCC) method, ER-immunocytochemical assay (ER-ICA) and ER-immunocytochemically stained 3H-thymidine autoradiography. In the tamoxifen (TAM) group and the medroxyprogesterone acetate (MPA) group, tumor growth was inhibited, but it was promoted in the 17-beta-estradiol dipropionate (E2) group. The ER level by the DCC method was significantly decreased in the TMA, the MPA and the E2 groups. The ER-ICA showed that the percentage of ER-positive cells was decreased in the TAM and the MPA group, but it was increased in E2 group. However, the ER-immunocytochemically stained 3H-thymidine autoradiography showed that not only the labelling index of ER-positive cells but also that of ER-negative cells was significantly decreased in the TAM and the MPA groups, while the labelling index was significantly increased in the E2 groups. Therefore, it was concluded that endocrine therapy affected the proliferation of both ER-positive cells and ER-negative cells of ER-positive breast cancer.     

Comparative studies of estrogen receptor determinations by enzyme immuno-assay using the monoclonal antibody, dextran-coated charcoal, and sucrose density gradient methods

Estrogen receptor (ER) determination in human mammary tumors was performed by enzyme immunoassay (EIA) using monoclonal antibody, and the results were compared with those obtained using the dextran-coated charcoal (DCC) and sucrose density gradient (SDG) method. Twenty ER-positive tumors by the DCC method were all ER-positive by EIA, when determined in the same cytosol fraction. Three out of 21 ER-negative tumors by the DCC method were ER-positive by EIA. Estrogen receptors of 8S, 8S + 4S, and 4S determined by the SDG method were all shown to be ER-positive using EIA. Between the values of estrogen receptors determined by the DCC method and by EIA, a high correlation was observed (r = 0.868). Values of ER less than 13.5 fmol/mg protein were considered as negative ER.     

Multiparametric study (SAMBA 200) of estrogen receptor immunocytochemical assay in 400 human breast carcinomas: analysis of estrogen receptor distribution heterogeneity in tissues and correlations with dextran coated charcoal assays and morphological data

Estrogen receptor (ER) immunocytochemical assay (ER-ICA) was assessed in 400 human breast carcinomas. In all cases, patient's age, tumor size, histological type and Scarff-Bloom-Richardson grade, and presence or absence of axillary lymph node metastases and of vessel invasion in tumor borders were recorded. In 310 cases estrogen and progesterone receptors were concomitantly evaluated (dextran coated charcoal method). In 60 of these cases the ER immunoenzymatic assay (ER-IEA) was also assessed. Monoclonal H222sp gamma and peroxidase antiperoxidase procedures (Abbott kit) were applied in frozen sections, tumor imprints, and fine-needle aspirates. A computerized system of image analysis referred to as SAMBA (TITN), permitted a multiparametric quantitative analysis of ER-positive surfaces. With this system, in each tumor, the cellularity, percentage of ER surface versus the total cell surface and versus the epithelial (keratin-positive) surface, integrated optical density, mean optical density, index of the concentration of labeled objects, and integrated optical density histograms, were obtained and correlated to histological and biochemical data. It was shown that (a) ER antigenic sites were heterogeneously distributed in ER-positive tumors, with a specific nuclear localization in epithelial cells; (b) SAMBA 200 multiparametric analysis of the ER sites distribution in tissue was appropriate, accurate, reproducible, and therefore more reliable than the semiquantitative analysis; (c) standardization and complete automation of this method of immunoprecipitates evaluation on tissue section permits daily and routine analysis of a large number of preparations; (d) there was a correlation between ER binding sites evaluation (dextran coated charcoal) and ER antigenic sites immunodetection (ER-ICA and ER-IEA); (e) there was a correlation between the SAMBA evaluation of ER-ICA and other histological prognostic factors such as small tumor size, low Scarff-Bloom-Richardson grade; (f) the preliminary SAMBA analysis of ER-ICA in tissue sections, imprints, and fine needle aspirates suggest that fine needle aspirates may not reflect accurately the tumor cell heterogeneity.     

DCC单点法及多点法测定乳腺癌雌 孕激素受体的比较研究

A comparative study of estrogen receptor (ER) and progesterone receptor (PgR) by single-point method and multi-point method in dextran coated charcoal assay (DCC) was carried out in 50 and 47 cases of breast cancer, respectively. Taking 10 fmol/mg protein as the positive value, the conformation rate of both methods in ER measurement was 96.0% (48/50) with r = 0.978 by linear regression analysis (P less than 0.001). In PgR measurement, the conformation rate of both methods was 95.7% with r = 0.988 (P less than 0.001). Statistically, the difference between single-point method and multi-point method in the ER and PgR measurements was not significant (P greater than 0.5) both in rank-sum test and in paired t-test. The authors suggest that ER and PgR of breast cancer samples be measured with single-point method for its simplicity and less tumor tissue required.     

Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early-stage breast carcinomas: Prognostic significance

The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunohistochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r = 0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P = 0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P = 0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P = 0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stromarich tumors, and early-stage lesions including intraductal carcinoma. © 1996 Wiley-Liss, Inc.     

Immunocytochemical assay of estrogen receptors in puncture products of breast carcinomas

In a pilot study, estrogen receptors (ER) were assayed on 42 surgically removed breast tumors by the following 3 methods: biochemical assay with dextran coated charcoal (DCC), Abbott immunoenzymatic (ER-EIA) and immunocytochemical (ER-ICA) technics. DCC and ER-EIA were performed on biopsy specimens while ER-ICA was run on cytocentrifugated cells obtained by fine needle aspiration (FNA). ER contents were expressed according to an index taking into account the proportion of colored neoplastic cells and the intensity of staining. Statistical correlation coefficient (Spearman and Kendall) concordance, sensitivity and specificity between the results were calculated (ER - ICA/ER - EIA: P less than 0.001, r = 0.38, concordance = 83%, sensitivity = 86%, specificity = 77%; ER - ICA/DCC: P less than 0.05, r = 0.22, concordance = 77%, sensitivity = 85%, specificity = 63%; ER - EIA/DCC: P less than 0.001, (r = 0.60). As previously reported, both immunoassays showed good agreement. The weaker but nevertheless significant correlation found with reference DCC may be due to the heterogeneity of tumoral ER content. This hypothesis is supported by the variability of ER - ICA assays on multiple FNA performed in 16 cases from our series. Use of multidirectional FNA slightly improved the results. Nevertheless, ER - ICA appear to be a good semi-quantitative method and might be helpful in the follow-up of metastasis treated with anti-estrogen, especially in small lesions not assayable by DCC.     

Hormone receptors and disease-free survival in breast cancer: impact of increasing threshold levels

Laboratory data from Milan and Houston were evaluated to determine the extent to which the distribution of estrogen receptor (ER) and progesterone receptor (PgR) has changed with time. Results from over 11,500 ER and over 8,200 PgR determinations (6,194 ER and 3,127 PgR from Milan) were analyzed. All assays in Milan were performed by a dextran-coated charcoal method and in Houston by a sucrose density-gradient method. The data demonstrate a time-dependent, upward drift in the amount of ER and PgR detected, with the effect most pronounced at the lower end of the distribution curves. We attribute this change to optimization of all facets of the receptor assay procedures (tissue harvesting and storage) as well as to a change in breast cancer biology. These results suggest that studies correlating certain biological parameters with receptor status (whether using qualitative or quantitative scales) need to be re-examined. For example, a population of 349 node-negative patients who did not receive any adjuvant treatment was studied in Milan to determine any association between disease-free survival (DFS) and receptor status. If the "historical" threshold values (10 fmol/mg protein) were used to determine receptor status, no significant difference in DFS at 5 years was detected. Even the combination of ER and PgR did not improve the predictive power of receptor status. In the premenopausal subgroup, ER status did predict the 5-year DFS. However, if the threshold value for PgR was adjusted to 25 fmol/mg protein, patients with ER-positive, PgR-positive tumors had significantly better 5-year DFS than patients with ER-negative, PgR-negative tumors. In addition, PgR status alone was associated with significantly improved 3-year DFS if the subgroups of PgR less than 5 fmol/mg protein and PgR greater than 100 fmol/mg protein were compared. We conclude from these data that: 1) historical threshold values for receptor positivity should be re-examined in all laboratories; 2) studies involving receptor results determined over an extended period of time should attempt to "normalize" these results; and 3) the quantitative assessment of receptor status should be used whenever possible.     

Estrogen and progesterone receptors as prognostic factors in breast cancer

The relation between estrogen receptors (ER) and/or progesterone receptors (PgR) and some clinical factors such as tumor size, axillary node involvement, histological tumor grade, and disease-free interval (DFI) in 500 patients with operable (TNM stage I-III) breast cancer was studied. ER-positive (ER+) tumors were commoner in older patients, whereas PgR-positive (PgR+) tumors were similarly distributed within the age groups. The concentration of ER+ protein also increased with age in contrast to PgR+ protein concentration. However, receptor status was not associated with menopausal status independently of age. Axillary node involvement influenced neither ER nor PgR status, but there was a statistically significant relation between tumor size and positivity of ER or PgR. There was no association between histologic tumor grade and either steroid receptor phenotype. DFI was longer in patients with ER+ than those with ER- tumors, independently of axillary nodal status. The positivity of PgR in patients with ER+ tumors contributed to an even longer DFI, suggesting that the combination of ER/PgR is a better indicator of DFI than ER or PgR alone.     

Prognostic value of estrogen and progesterone receptors measured by enzyme immunoassays in human breast tumor cytosols

Clinically significant cut-off values to discriminate between receptor-positive and -negative, and the prognostic value of estrogen receptors (ER) and progesterone receptors (PgR) measured by enzyme immunoassay (EIA) have not yet been established. We have therefore measured ER and PgR by EIA in cytosols from 205 primary breast cancer biopsies. Clinically significant cut-off values (30 fmol/mg protein for ER; 27 fmol/mg protein for PgR), as related to tumor recurrence (median follow-up, 47 months), have been established by isotonic regression analysis. These data were compared to those obtained by simultaneously performed dextran-coated charcoal (DCC) assays (cut-off values: 18 fmol/mg protein for ER, and 26 fmol/mg protein for PgR) on the same cytosols, and to DCC assays performed previously (up to 10 years ago) on cytosols prepared from other parts of the tissue biopsies (cut-off values: 18 fmol/mg protein for ER, and 23 fmol/mg protein for PgR). Using the cut-off values for the EIA and the DCC assays performed on the same cytosols, the discrepancies between receptor status appeared less than 10% both for ER and for PgR. Furthermore, the concentrations of ER or PgR detected with the EIA or DCC assay were highly and significantly correlated (Spearman rank correlations: for ER, Rs = 0.94; for PgR, Rs = 0.88; P less than 0.0001). After classification in different phenotypes with respect to ER/PgR status (+/+, +/-, -/+, and -/-), analysis for relapse-free survival and overall survival showed equal prognostic power in the comparable groups in the order, from favorable to unfavorable, of +/+ greater than +/-(-/+) greater than -/- (chi2: P less than 0.0001), irrespective of the assay which has been used for quantification of the receptor. It is concluded that both the conventionally used DCC and the newly available EIA methods are equally useful for assessing ER and PgR status.     

Distribution of estrogen and progesterone receptors in primary tumor and lymph nodes in individual patients with breast cancer

Primary breast cancer tissue and lymph nodes were obtained from 48 patients. Estrogen receptors (ER) and progesterone receptors (PgR) were determined by a dextran-coated charcoal assay. ER were present in 72.9% of the primary tumors and in 62.4% of the malignant lymph nodes, whereas PgR were present in 73.0% and 50.0% of the cases, respectively. The primary tumor and the corresponding malignant lymph nodes showed an identical ER and PgR status, i.e., both tumor sites were receptor positive or both receptor negative in 89.6% and 77.1%, respectively. However, 10.4% of the patients had ER-positive tumors but ER-negative lymph nodes and 22.9% had PgR-positive primaries with PgR-negative lymph nodes. No receptor-positive lymph nodes showed a combination with receptor-negative primary tumor. This preliminary data shows that receptor-positive malignant lymph nodes mostly display the same receptor status as the corresponding primary tumor, whereas receptor-negative lymph nodes may have a receptor-positive primary tumor.     

Progesterone receptor in hepatocellular carcinoma. Correlation with androgen and estrogen receptors.

Progesterone receptors (PgR), estrogen receptors (ER), and androgen receptors (AR) were assayed consecutively for hepatocellular carcinoma (HCC) that was surgically removed from 19 men and three women. The methods of receptor assay were the enzyme immunoassay (EIA) for PgR and the dextran-coated charcoal (DCC) technique for ER and AR. The patients ranged in age from 32 to 77 years (average, 60.3 years). No patients had received any specific anti-cancer therapy before tissue collection. All patients but one had underlying liver disease: cirrhosis in 13 and chronic hepatitis in eight. The positive rate of each receptor was 18% for PgR, 48% for ER, and 82% for AR. The titer was highest for AR, intermediate for ER, and lowest for PgR. The titers of PgR in four PgR-positive patients ranged from only 1.1 to 3.0 fmol/mg of protein. There was no relationship between PgR, ER, and AR in terms of positivity and titer. Also, other clinical and histopathologic data did not influence the positivity or concentration of these three sex hormone receptors. It can be concluded that no or little PgR exists in the cytosol of untreated HCC.     

Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases.

PURPOSE: To determine whether progesterone receptor (PgR) status provides additional value to estrogen receptor (ER) status and improves prediction of benefit from endocrine treatment among patients with primary breast cancer. PATIENTS AND METHODS: Clinical outcomes of patients in two large databases were analyzed as a function of steroid receptor status. The first database (PP), contained 3,739 patients who did not receive any systemic adjuvant therapy and 1,688 patients who received adjuvant endocrine therapy but no chemotherapy. The second database (SPORE), contained 10,444 patients who received adjuvant endocrine therapy but no chemotherapy. Biochemical ER and PgR assays were identically performed in two different central laboratories. RESULTS: In univariate and multivariate analyses, the prognostic significance of PgR status among systemically untreated patients is modest. Among endocrine-treated patients, however, multivariate analyses, including lymph-node involvement, tumor size, and age, demonstrate that PgR status is independently associated with disease-free and overall survival. For recurrence, the reduction in relative risk (RR) was 25% for ER-positive/PgR-negative patients and 53% for ER-positive/PgR-positive patients, compared with ER-negative/PgR-negative patients (P <.0001, PP patients). Patients with ER-positive/PgR-negative tumors have a reduction in RR of death of 30% (SPORE patients) and 38% (PP patients), compared with patients with ER-negative/PgR-negative tumors (P <.0001). For ER-positive/PgR-positive tumors, the reduction of the risk of death was greater than 46% in SPORE patients and 58% in PP patients, indicating that ER-positive/PgR-positive patients derive more benefit from endocrine therapy (P <.0001). CONCLUSION: When accurately measured, PgR status is an independent predictive factor for benefit from adjuvant endocrine therapy. Therefore, PgR status should be taken into account when discussing RR reductions expected from endocrine treatment with individual patients.     

Immunoperoxidase localization of estrogen receptors in human brest carcinoma

Monoclonal antibodies to the estrogen receptor protein were used to evaluate the specificity of the peroxidase antiperoxidase (PAP) method for localization of estrogen receptors (ER) in human breast carcinoma. Twenty-five cases of breast carcinoma were selected, and they were roughly classified as ER-rich and ER-poor tumors based on ER levels as determined by dextran-coated charcoal (DCC) assay. ER-rich tumors showed strongly positive cytoplasmic and, to a lesser degree, nuclear staining. Both the cytoplasmic and nuclear staining were less intense in ER-poor tumors, and the staining intensity varied from one to another cell population in the same tumor. Completely negative cells were also observed. The results of the PAP and DCC methods correlated fairly well in ER-rich tumors but not in ER-poor tumors. The PAP method provided visual localization of ER combined with excellent tissue morphology. Application of both methods (DCC and PAP) may provide better assessment of the estrogen receptor levels in breast carcinomas.     

Detection of estrogen receptor in bone marrow from patients with metastatic breast cancer

We devised a method of detecting estrogen receptors (ER) in bone marrow metastases from patients with breast cancer. The method involves a sequential double-staining immunocytochemical technique, with a monoclonal antibody to ER and a polyclonal antibody recognizing epithelial membrane antigen to confirm the epithelial nature of suspected tumor cells. Twenty-seven patients were assessed: ten were found to have ER-positive tumor cells in the bone marrow; ten had ER-negative cells; and the remaining seven patients had no tumor cells in the bone marrow smears. Of the ten patients with ER-positive cells, eight (80%) either had a response to endocrine therapy, implying that they possess ER-positive breast cancers, or had ER-positive primary tumors as determined by the dextran-coated charcoal biochemical assay (DCC). Of the ten patients with ER-negative cells in the bone marrow, eight failed to respond to endocrine therapy. This technique therefore provides a means of predicting which patients will respond to endocrine therapy, and is particularly important in those patients whose ER status is unknown.     

Estrogen receptors in human gastric, hepatocellular, and gallbladder carcinomas and normal liver tissues

Steroid binding assay using the dextran coated charcoal (DCC) method was applied to human tissues including tumors of the digestive organs, and the results were compared with those of enzymeimmunoassay (EIA) and immunocytochemical assay (ICA) with monoclonal antibody against human estrogen receptor of MCF-7 breast cancer cells. Using the DCC method, estrogen receptor activity was detected in 6 of 26 cases (23.1%) with gastric carcinoma, 3 of 16 hepatocellular carcinoma cases (18.8%), 1 of 3 gallbladder carcinoma cases (33.3%), and both of the 2 cases (100%) with normal liver tissue. However, using EIA, no ER activity was detected in any case. Moreover, ER positive cells were not found by immunohistochemical staining in the gastric carcinoma cases or in normal liver tissue, both of which showed ER activity by the DCC method. These results suggest that the estrogen receptor like material exists in cytosol of the human digestive tumors and normal liver tissue, but that the specificity of the antibodies against estrogen receptor molecules in these tumors may be different from that of the breast tumors.