Monoclonal antibodies for progesterone receptor assays and polymorphism studies in breast cancer. Comparison with radioligand assays

Progesterone receptors (PR) from human breast tumors were assayed by a new method using monoclonal antibodies immobilized on beads (PR-EIA, Abbott Laboratories). EIA results were compared to those obtained with the dextran-coated charcoal method using a tritiated ligand (ORG 2058). The precision and reproducibility of the EIA method were studied over a 3-month period: intra-assay coefficients of variation were less than 6% for the range of the assay (between 5-250 fmol/ml), and inter-assay coefficients of variation calculated on 13 consecutive standard curves were less than 10%, except for standard 0 (33%). PR assays performed on 78 tumors both by EIA and radioligand (RLA) were compared. The linear regression obtained was: PR-EIA = 0.81 RLA+1 fmol/mg protein (r = 0.88). For reproducibility studies, cytosols were assayed twice during a period ranging from 1 week-3 months, both by EIA and RLA. The linear regression obtained between the second assay (B) and the first assay (A) was: B = 0.98 A + 11 fmol/mg protein for RLA (r = 0.98), and B = 0.99 A-7 fmol/mg protein for EIA (r = 0.98). To study the effect of KCl on PR-EIA, 26 tumors were homogenized in 0.4 M KCl Tris buffer and assayed both by EIA and RLA. A good correlation was obtained between the 2 methods, but higher values were obtained with PR-EIA (P = 1.6) in comparison with RLA. The addition of KCl to the cytosol showed that KCl had no effect on EIA results, but significantly lowered RLA results. To study the effect of KCl on progesterone receptor isoforms, cytosols were analyzed by chromatography on TSK G3000 SW columns, and the presence of PR was detected in each fraction by both EIA and RLA. In the absence of KCl, only the oligomeric form of PR was observed; however with both techniques, detection of this form different from tumor to tumor, emphasizing the inter-tumoral molecular heterogeneity of PR. After PR isoform dissociation by KCl (0.4 M) and chromatographic analysis of the forms obtained, monoclonal antibodies detected PR molecular forms different from those observed by radioligand; furthermore, chromatographic patterns obtained were different from one tumor to another and confirmed the inter-tumoral molecular heterogeneity of the progesterone receptor.     

Comparison of ligand binding assay and enzyme immunoassay of oestrogen receptor in human breast cancer cytosols

Summary Nine laboratories cooperating in the Receptor Study Group of the EORTC compared results of ligand binding assay supplemented with Scatchard plot analysis (LBA) and enzyme immunoassay (EIA) for the assessment of oestrogen receptors in 1665 breast cancer cytosols. An excellent agreement was observed between results of Scatchard plot analysis and EIA in each laboratory. Linear correlation coefficients of log-transformed data (log [(ER + 10)/10]) ranged from 0.839 to 0.977 (n = 52–373; P < 0.001); Spearman's R ranged from 0.797 to 0.972 (P < 0.001). Orthogonal regression analysis on log-transformed data revealed slopes of 0.794 to 1.141 and intercepts of – 0.057 to 0.154 corresponding to – 1.2 to 4.3 fmole/mg protein. Both assays compared equally well for pre- as well as postmenopausal patients, which confirms that occupied receptors are not extracted during the preparation of cytosol. The percentage discordance in the classification of tumours as ER positive or negative varied from 4.1 to 13.3 when a cut-off value of 10 fmol/mg protein was used, and from 1.4 to 7.5 at a cut-off level of 20. Considerable variations were observed in the actual receptor levels reported by each laboratory. Since these differences occurred in the results of both methods, they are attributed to differences in tissue handling. It is concluded that the ER-EIA is an excellent alternative to Scatchard plot analysis for the assay of oestrogen receptors. It is recommended, however, that laboratories performing assays on samples obtained from patients who are eligible for entrance in EORTC clinical trials and who wish to use the EIA should validate this assay against the Scatchard plot assay in their own setting.     

Steroid receptor measurement in breast cancers: Comparison between ligand binding and enzyme-immunoassay in cytosolic and nuclear extracts

We have analysed cytoplasmic and nuclear extracts of breast-cancer tissue from a total of 799 patients, measuring both oestrogen and progesterone receptors (ER, PR) using either the ligand binding assay (LBA) or the enzyme immuno-assay technique (EIA). Mean and median receptor levels were much lower than those widely reported by others. For ER, this may in part be a consequence of the younger median age of the patient group. The frequency of positivity, using consensus cut-off values for clinical evaluation, was also lower than that reported by the EORTC Receptor Study Group. Although the measurements comparing the 2 methods were statistically correlated in terms of positivity, based on the above criteria for clinical assessment, concordance was considered to be relatively poor, particularly for ER when assayed in the same samples by the 2 methods. In cytosolic but not nuclear extracts, the LBA method gave a higher median value for ER than the EIA (except in the group that had EIA values greater than 15 smol/mg protein); for PR, median values were higher with EIA in both cell fractions. There was an excellent correlation between receptor amounts in cytosolic and nuclear extracts for both ER and PR using the EIA; this was significantly better than with LBA. We also observed a correlation between ER and PR in both cytosolic and nuclear fractions which was most pronounced when the analysis was done by EIA. The amounts of ER in the cytosolic fraction were also correlated with the those of PR in the nuclear fraction and ER in the nuclear fraction with PR in the cytosolic fraction, but only when the EIA method was used. We conclude that the EIA method appears to be more sensitive and gives biologically more reliable results. However, the disagreement between the methods may be due to legitimate recognition of altered forms of the receptor and may be of biological significance. Although the presence of receptor in the cytosolic fraction is artifactual, its measurement by EIA does parallel the amounts of nuclear receptor, which may be a more relevant biological parameter. Int. J. Cancer 71:526-538, 1997. © 1997 Wiley-Liss, Inc.     

Cell cycle expression of steroid receptors determined by image analysis on human breast cancer cell line: A hypothesis on the effects of antiestrogens

Tamoxifen is the widespread anti-hormonal compound used for the treatment of human breast cancer. It is admitted that its effects are mediated via estrogen receptors (ER) but the molecular basis of its activity has yet to be clearly defined. In this work, we have developed a new image cytometry procedure in order to clarify the interactions between steroid receptors and tamoxifen at the cell cycle kinetic level. On untreated cells, an increase of ER level and a decrease of progesterone receptor (PR) level during the G0/G1 phase were demonstrated. Then, the ER and PR levels fell during the S-phase until the beginning of G2/M phase, where an increase was observed, especially for PR. These results suggest that ER is synthesized preferentially during the G0/G1 transition and PR during the S/G2 transition. After short-term tamoxifen treatment an augmentation of ER level was observed which was not dose-dependent, suggesting an increase in receptor translation rather than an augmentation of ER synthesis. PR level declined in the majority of the population leading to a selection of a subset of proliferating PR negative cells after treatment. These data demonstrate that the synthesis of steroid receptors is linked with the progression of cells through the cell cycle and indicate that tamoxifen blocks MCF-7 cells in G1 via its interactions with ER. Our multifluorescence imaging procedure appears to provide a rapid and quantitative approach which is useful for investigating alterations in steroid receptors after endocrine treatment.     

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.     

乳腺癌ER、PR、HER-2在原发灶及复发转移灶中表达变化

目的：探讨ER、PR及Her-2在乳腺癌原发和复发转移灶中的表达变化及其相关性。方法：免疫组化法检测45例乳腺癌原发及复发转移灶中的ER、PR及HER-2表达。结果：ER在原发灶和复发转移灶之间的变化率为66.67%（30/45）,PR总的变化率为17.78%,cerbB2癌基因蛋白总的变化率13.33%。结论：ER在乳腺癌原发灶和复发转移灶之间的表达的差异具有统计学意义（P〈0.05）。PR及cerbB2癌基因蛋白的表达在原发灶和复发转移灶之间差异无显著性（P〉0.05）。     

A quality control study to assess the inter-laboratory variability of routine estrogen and progesterone receptor assays

The steroid hormone receptor laboratories of Bern, Basel, Zürich, Locarno, Lausanne and Geneva have participated in a quality control study to assess potential inter-laboratory variability in results of tests for determination of steroid hormone receptor status in human breast tumor biopsies. Homogeneous breast tumor powders containing low, medium and high concentrations of estrogen receptors (ER) and progesterone receptors (PR) were prepared in Bern and dispatched on solid CO₂ to each laboratory within 1 day of preparation. Each laboratory was requested to assay each powder for ER and PR by their usual procedures. The results revealed that the quantitative discrepancies in ER and PR binding values among the participants could be attributed in part to variations in the methods used for measuring cytosol protein content and also to the differences in hormone receptor assay methods. Nevertheless, all of the laboratories were able to identify the samples containing low, medium and high concentrations of ER and PR.     

Measurement of estrogen receptor and progesterone receptor in human endometrium and endometrial carcinoma

The concentration of cytosol estrogen receptors (ER) and progesterone receptors (PR) in the endometrium of the normal menstrual cycle and endometrial carcinoma, were measured by Enzyme Immunoassay (EIA) using monoclonal antibody, and were compared with Dextran Coated Charcoal (DCC) method. In DCC method, maximum binding sites were estimated according to Scatchard plot analysis. Following results were obtained in this study. 1) In the normal endometrium obtained from 20 cases, the correlation coefficients for ER and PR were 0.907 and 0.778, respectively. Regression lines were as follows; ER (EIA) = 1.68 (DCC) + 19.1 fmol/mg protein and PR (EIA) = 0.13 (DCC) + 24.8 fmol/mg protein. A good correlation was found between the two methods in ER assay. 2) In the normal menstrual cycle, DCC values and EIA values of ER were increased in proliferative phase, and were decreased in secretory phase. DCC values of PR were increased in proliferative phase and not decreased in secretory phase, but EIA values of PR were not remarkably changed. 3) In the endometrial carcinoma obtained from 14 cases, there was good correlation between EIA and DCC values in ER assay (r = 0.941), but correlation between the two methods was not found in PR assay. 4) In relation of histology, positive rates were highest in patients with well differentiated types, and in relation of clinical stage, positive rates were higher in the patients with early stages than progressive stages. These results suggest that EIA is as useful as DCC in ER assay in normal endometrium and endometrial carcinoma.     

Determination of estrogen and progesterone receptors in endometrial adenocarcinomas. Comparison between dextran-coated charcoal and immunoenzymatic methods on curettage biopsies.

Estrogen receptors (ER) and progesterone receptors (PR) were determined on curettages from women with endometrial adenocarcinoma. The results obtained with the enzyme immunoassay (EIA) and the dextran-coated charcoal (DCC) assay were compared. A highly significant correlation was obtained between these methods for the ER measurement (Rs = 0.91). For PR determination, the Rs value between EIA and DCC assay was 0.57 and the mean value of PR-DCC is significantly higher than the mean value of PR-EIA. These results suggest that EIA is a suitable method for ER measurement. For PR determination on curettage material the DCC assay seems more accurate than EIA.     

Discordant results between radioligand and immunohistochemical assays for steroid receptors in breast carcinoma

Surgical biopsy specimens of 179 breast carcinoma were studied by steroid-binding and immunohistochemical assays or oestrogen and progesterone receptors (ER, PR) in order to explore reasons for discordant results between the two assay types. Receptor statuses in 18% of ER assays and 30% of PR assays were in disagreement. Immunohistochemistry-positive steroid-binding-negative status predominated among the discordant ER assays, while the discordant PR assays displayed the opposite situation. In discordant assays receptor concentration was significantly more often close to the cut-off (10-50 fmol mg-1) than in the concordant ones. Low binding affinity (high Kd) was also significantly associated with disagreeing assay results. These observations clearly indicate that immunohistochemical ER and PR assays measure high-affinity binding components (i.e. type I receptors) in steroid-binding assays. ER but not PR assays in premenopausal women disagreed more often than those in post-menopausal women. Such factors as histological type, specimen size in steroid-binding assay, grade of malignancy and tumour necrosis were statistically unrelated to agreement or disagreement of receptor assays.     

子宫内膜癌雌、孕激素受体和C-erbB-2检测的临床意义

目的 检测子宫内膜癌组织中雌激素受体(ER)、孕激素受体(PR)及癌基因蛋白C-erbB-2表达的阳性率并探讨其与预后的关系.方法 用免疫组织化学法对32份子宫内膜癌标本进行了ER、PR及C-erbB-2的检测.结果 子宫内膜癌组织中ER、PR、C-erbB-2的阳性率分别为53.1%、50.0%、46.9%.ER、PR的阳性表达率与癌组织的细胞分化程度有关,随着子宫内膜癌组织学分级的增高,ER、PR阳性表达率逐渐降低,C-erbB-2的阳性表达率与肿瘤病理分级呈正相关,与ER、PR表达呈负相关.结论 ER、PR、C-erbB-2均反映了子宫内膜癌的生物学行为,其测定对预测预后、指导选择内分泌治疗具有重要意义.     

Immunohistochemical assessment for estrogen receptor and progesterone receptor status in breast cancer: Analysis for a cut-off point as the predictor for endocrine therapy

BACKGROUND: An immunohistochemical (IHC) method is commonly used for determining estrogen receptor (ER) and progesterone receptor (PR) status in breast cancer. However, the proper cut-off points of IHC have not been established. Cut-off points for ER and PR status as predictive factors for endocrine therapy are needed. METHODS: A total of 249 cases of female breast cancer were enrolled. ER and PR status by IHC were analyzed using the proportion of stained cells and staining intensity by Allred's score. RESULTS: Proportion score (PS) and intensity score (IS) were related to enzyme immunoassay (EIA) titers, for both in ER and PR (p < 0.0001, all). PS correlated with IS in both ER and PR (R = 0.47 and 0.41, respectively). ER status by IHC was related to tumor size and lymph node status, while PR was related to tumor size and menopausal status. In 152 patients who received endocrine therapy with a median follow-up term of 38 months, differences in disease-free survival were most significant using a cut-off point of PS 3 which indicated more than 10 % of cells stained positively for both ER and PR (p = 0.0007 and 0.0087, respectively). In addition, combination analysis of ER and PR using this cut-off point revealed a notable prognostic difference. CONCLUSION: A 10 % staining proportion may be an acceptable cut-off point for both ER and PR status by IHC, in terms of predicting response to endocrine therapy in breast cancer.     

乳腺癌肿瘤组织不同部位雌孕激素受体的表达

目的 探讨乳腺浸润性导管癌肿瘤组织中不同部位雌、孕激素受体表达情况.方法 收集32例手术切除乳腺浸润性导管癌肿瘤标本,于每例肿瘤标本4个不同部位取材,用免疫组化方法检测各部位雌、孕激素受体表达情况.结果 肿瘤组织不同部位雌、孕激素受体检测结果一致性好,最好Kappa值分别为0.789和0.810,最差Kappa值分别为...     

结直肠癌雌激素受体及孕激素受体的定量研究

背景与目的许多研究表明结直肠癌与雌激素相关,结直肠癌组织中表达雌激素受体(estrogenreceptor,ER)、孕激素受体(progesteronereceptor,PR),但多采用免疫组化方法检测,且结果不一,而ER.PR的定量研究不多。本研究拟定量测定结直肠癌组织及正常组织的ER,PR表达水平,并探讨它们之间的关系及与结直肠癌患者临床病理参数的关系。方法应用受体放射配基结合分析法(radioligandbindingassay,RBA)定量测定45例结直肠癌组织及结直肠正常组织的胞浆及胞核ER、PR的水平。结果45例标本结直肠正常组织及癌组织都表达ER、PR,胞浆ER表达癌组织高于正常组织,分别为7.96±3.69fmol/mgprotein和4.34±2.84fmol/mgprotein,(P<0.01);胞浆PR表达癌组织高于正常组织,分别为3.89±2.64fmol/mgprotein和2.50±1.73fmol/mgprotein,(P<0.01);胞核ER表达癌组织高于正常组织,分别为18.42±8.30fmol/mgprotein和11.24±5.44fmol/mgprotein,(P<0.01);胞核PR表达癌组织高于正常组织,分别为9.36±5.90fmol/mgprotein和7.84±7.41fmol/mgprotein,(P<0.05)。癌组织胞浆及胞核的ER表达与PR表达呈正相关,(P<0.01);正常结直肠组织胞浆ER表达与PR表达呈正相关,(P<0.01),而胞核内两者不相关,(P>0.05)。结直肠癌组织ER表达与患者年龄相关,大于45     

Breast cancers negative for estrogen receptor but positive for progesterone receptor, a true entity?

By the conventional steroid-binding assay method for receptor, 3% of 1,095 primary breast cancers (or 10.6% of 263 premenopausal tumors) were classified as negative for estrogen receptor (ER), but positive for progesterone receptor (PR). The true ER status in this rare group of tumors was further investigated by the enzyme-immunoassay (EIA) or immunocytochemical (ICA) staining method using monoclonal antibodies H222 and D547. Immunoreactive ER was present in nine ER-/PR+ tumors studied, whereas it was not detectable in nine age-matched ER-/PR- tumors. Immunoreactive ER was also present in 24 ER+ breast cancers studied, and was particularly higher in tumors that were PR+. Measurement of immunoreactive ER by monoclonal antibody method provides certain advantages over the conventional dextran-coated charcoal (DCC) method, especially in ER-/PR+ tumors.     

Influence of endocrine status on biochemical and immunocytochemical estrogen and progesterone receptor assays in breast cancer patients

Summary Breast cancer tissue from 190 patients was studied for immunocytochemically reactive estrogen and progesterone receptors (ER, PR). Parallel cytosol ER and PR assays were performed on 159 of these patients using the dextran-coated charcoal (DCC) method. For the immunocytochemical determination, monoclonal antibodies to ER (ER-ICA kit) and PR were used in an immunoperoxidase procedure. Agreement between the two techniques in postmenopausal patients was better than in the premenopausal group (ER, kappa = 0.597 vs. 0.398; PR, kappa = 0.460 vs. 0.329). The median ER cytosol concentration in receptor-positive postmenopausal patients was significantly higher than in receptor-positive premenopausal patients (87 vs. 31 fmol/mg cytosol protein, p<0.001). A similar trend was also found in the immunocytochemical ER assay (270 vs. 207 histoscore units, p>0.05). Significantly higher cytosol ER contents were found in patients with low serum estradiol concentration. The proportion of ER-negative tumors was slightly higher in the premenopausal patients by both methods. In the PR assays (biochemical or immunocytochemical) there were no significant differences between the two patient groups in the proportion of PR-negative tumors or in the median PR content in PR-positive tumors.     

Total prolactin binding sites in human breast cancer biopsies

Summary Total and free prolactin receptors were assayed in 72 human breast tumor biopsies. Forty-nine percent of the tumors are positive (specific binding greater than 0.8% of total radioactivity) when assaying free receptors, while 71% are positive when total receptors levels are determined. No clear relationship exists between the prolactin receptor positivity and the presence of either progesterone or estradiol receptors. Measurement of total prolactin receptors could be an important and independent criterion of the hormonal sensitivity of the tumors. Address for reprints: Dr. J.P. Peyrat, Laboratoire d'Endocrinologie Expérimentale, Centre de Anticancéreux de Lille (Centre Oscar Lambret), BP 307, 59020 Lille Cédex, France.