Susan M. Thorpe

The prognostic value of immunohistochemical estrogen receptor analysis in paraffin-embedded and frozen sections versus that of steroid-binding assays

Estrogen receptors (ER) were independently analyzed using dextran-coated charcoal assays (ER-DCC) and immunohistochemical assays in frozen (ER-ICA) and paraffin-embedded tissue (ER-PAR) from 130 human breast cancer specimens drawn from postmenopausal high-risk patients registered in the Danish Breast Cancer Cooperative Group. ER was best detected with the ER-DCC assay followed by the ER-ICA (relative sensitivity 87%) and the ER-PAR assays (relative sensitivity 71%). The semiquantified staining features of the immunohistochemical assays were statistically significantly correlated with each other and with ER-DCC. Analysis of disease-free interval (DFI) and overall survival (OS) showed that all assays allowed statistically significant discrimination between a high risk and a low risk group, although the sensitivity differences tended to be reflected as small differences in clinical discriminatory power. The patient groups were then stratified according to adjuvant treatment [radiotherapy (RT) versus radiotherapy and tamoxifen (RT + TAM)]. The survival advantage was tied primarily to the receptor status itself in the steroid-binding assays, but was linked to both the receptor status and the adjuvant treatment in the immunohistochemical assays. Thus, the relative risks in terms of DFI and OS were of the same relative magnitude in the RT and RT + TAM groups for ER-DCC assays using a cut-off level of 10 fmol/mg cytosol protein, while there were large differences in the relative risks between RT and RT + TAM groups for ER-ICA and ER-PAR assays. We conclude that an ER assay in fresh tissue should be given first priority, but if there is no fresh tissue, an ER assay in paraffin-embedded tissue offers a reasonably good alternative as a prognosticator and an equivalent alternative as a predictor of the response to endocrine treatment.

Short recurrence-free survival associated with high oestrogen receptor levels in the natural history of postmenopausal, primary breast cancer

The ability of oestrogen and progesterone receptor (ER and PgR, respectively) status to discriminate recurrence-free survival (RFS) among a cohort of consecutively accrued 952 postmenopausal patients has been studied. None of the cohort members investigated were treated with adjuvant therapy. Using a graduated scale of receptor status [low, intermediate and high receptor levels (< 10 vs. 10-107 vs. > or = 108 fmol/mg cytosol protein, respectively)] instead of the more commonly used dichotomous subdivision (positive vs. negative), ER level significantly discriminated between groups of patients with long vs. short RFS. Contrary to our expectations, patients with highest ER levels have as poor a prognosis as ER-negative patients, while patients with intermediate ER levels have longest RFS. The group of patients with ER levels > or = 108 fmol/mg cytosol protein comprises 47% of the cohort. The independent significance of overexpression of ER as a prognostic factor among this patient group is demonstrated in multivariate analysis where ER level is more significant than either grade of anaplasia or tumour size. PgR status did not significantly predict RFS among these patients. While the highest ER levels predispose for poorer prognosis among postmenopausal patients, it is precisely this group that experiences greatest benefit from adjuvant treatment with tamoxifen. Thus, patients who might otherwise go untreated due to their node-negative status can be readily identified and offered adjuvant treatment.

Immunocytochemical detection of progesterone receptor in breast cancer with monoclonal antibody: relation to biochemical assay, disease-free survival, and clinical endocrine response

A new immunocytochemical assay for progesterone receptor (PgR‐ICA) employing the monoclonal antibody JZB 39 was used to study tumors from two series of patients with breast cancer. In Series 1 assay results were in agreement with those of biochemistry in 76% of 338 cases (P < 0.001) and in 54% of 101 cases in Series 2 (P < 0.001). Agreement was better in Series 1 because it included fresher, previously untouched specimens. There were 70 patients in Series 1 with known clinical endocrine response. A negative assay correlated with disease progression in 45 of 57 patients, significantly better than with biochemistry (P = 0.013). In comparing 39 women with rapid disease progression with 39 free of disease at 5.1 years, those with PgR‐ICA‐positive tumors were over four times more likely to remain disease‐free than those with negative results (P = 0.007). Product moment life‐table analysis of 79 patients from Series 2 showed a significantly better cumulative survival for those with PgR‐ICA‐positive tumors (P = 0.047). These findings indicate that PgR‐ICA should be of value in planning therapy and predicting disease course in breast cancer patients.

Steroid receptors in breast cancer: sources of inter-laboratory variation in dextran-charcoal assays.

SummaryThe presently recognized correlations between various clinical parameters and the concentrations of estrogen and progesterone receptors in breast cancer biopsies are largely based on receptor values obtained using the dextran-coated charcoal (DCC) method. This assay method is highly sensitive to slight changes in assay protocol, and differences in assay methodology may account for the wide variation in proportions of receptor positive patients reported by different centers.A survey of various aspects of the assay method that may lead to reproducible, systematic differences in concentrations of receptor levels is presented; and methods of compensating for or correcting these potential differences are discussed. The following aspects are considered: a) constitution of biopsy tissue, b) method of tissue homogenization, c) adsorption of ligands to surfaces, d) inclusion of molybdate in the assay buffer, e) composition of the DCC slurry, and f) handling of samples for liquid scintillation counting.Differences in methods used to homogenize tissue in Europe and the U.S.A. may account for differences observed in the correlation of DCC assay results to results obtained using the recently-introduced monoclonal ER-EIA technique.

Reproducibility of Subjective Immunohistochemical Estrogen- and Progesterone Receptor Determination in Human Endometrium

To obtain a reliable scoring system for semi-quantitation of estrogen- (ER) and progesterone receptors (PgR) in human endometrial tissue, we investigated the reproducibility of subjective immunohistochemical ER and PgR determination. Specimens of frozen endometrial tissue were stained once (n = 129) or twice (n = 22) using the ER-ICA and PgR-ICA kits from Abbott. The semi-quantitative approach we used included subjective estimates of the overall staining intensity (I) and of the fraction of stained cells (%). Scoring was performed twice by the same observer and once by another observer (n = 87). Intra- and inter-observer agreement were evaluated using Kappa statistics. We found that more comprehensive scorings of I and % could not be agreed upon by the observers. Only simplified estimates of the fraction of cells stained and overall staining intensity were reproducible. Subjective estimates obtained by this method agreed with estimates obtained by counting (n = 38). Simplified H-scores, which were obtained by multiplication of the simplified estimates of % and I, were reproducible, too. In addition, semi-quantitation of ER and PgR by immunohistology was significantly correlated to quantitation by enzyme-immunoassay (n = 39). Thus, it was possible to reproducibly semi-quantitate ER and PgR only by employing a very simple immunohistochemical scoring of ER and PgR.

Antiestrogen treatment of postmenopausal women with primary high risk breast cancer

SummaryThe role of antiestrogen treatment in high risk postmenopausal patients with primary breast cancer is currently evaluated in a nationwide, prospective randomized trial conducted by the Danish Breast Cancer Cooperative Group. The primary treatment is total mastectomy and radiotherapy. As of February 1, 1982, 720 women were randomized to treatment with tamoxifen (30 mg daily for 1 year) and 691 women were randomized to no further therapy. Life-table analysis after 36 months shows a difference in recurrence rates of 9% (p = 0.19) in favor of the tamoxifen-treated patients. The material has been analyzed with respect to established prognostic factors such as age, degree of anaplasia, tumor size, and number of positive nodes. The rates of recurrent disease are lower in all subsets of patients treated with tamoxifen, but are only statistically significant in patients 50–59 years of age or with 4 or more positive lymph nodes. Regardless of treatment, ER negative patients have a 23% higher recurrence rate than ER positive patients after 18 months of analysis (p = 0.0033); this represents an approximate doubling of risk, and is independent of age, degree of anaplasia, tumor size, or lymph node status. With regard to PgR status, there is 11% higher recurrence rate in the PgR negative than in the PgR positive patients (p = 0.097).

Monoclonal antibodies applied to primary human breast carcinoma: Relationship to menopausal status, lymph node status, and steroid hormone receptor content

SummaryMonoclonal antibodies were raised against purified human milk fat globule membranes. The binding of three of these (Mam-3, HMFG 1, and HMFG 2) to fixed histological sections of 100 primary breast carcinomas has been investigated. Tissue specificity was investigated by testing the binding of the antibodies to carcinomas from other organs and to benign proliferative breast lesions.In breast tissue, antigens were found only in the epithelial cells, while the stromal component, myoepithelium, and any inflammatory cells present were negative. The reaction was characterized by considerable heterogeneity, both with regard to the percent of cells positive and the intensity of the reaction. No relationship was observed between binding and histological type of breast carcinoma. However, in ductal infiltrating carcinomas, a tendency towards a greater proportion of tumors that bound the antibodies was observed among the highly differentiated compared to the low differentiated carcinomas.Data on estrogen and progesterone receptors were available in 53 and 22 of the 100 carcinomas, respectively. There were no apparent relationships between the presence of surface antigens and the menopausal status, lymph node status, or estrogen receptor status, but the tumors lacking progesterone receptor apparently lacked the antigens as well.The presence of surface antigens probably defines a differentiation in primary breast carcinoma. Whether this differentiation is of any prognostic significance remains to be established.

Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor.

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis.

Synthesis and estrogen receptor binding affinities of novel pyrrolo[2,1,5-cd]indolizine derivatives

A series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor. Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their structural resemblance to estrogen.

Synthesis and pharmacological evaluation of novel cis-3,4-diaryl-hydroxychromanes as high affinity partial agonists for the estrogen receptor.

The syntheses and in vitro pharmacological evaluation of a number of cis-3,4-diaryl-hydroxy-chromanes are reported, along with the results of a thorough in vivo profiling of the tissue-selective estrogen partial-agonist NNC 45-0781 [3, (-)-(3S,4R)-7-hydroxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane]. These studies showed that NNC 45-0781 is a very promising candidate for the prevention of post-menopausal osteoporosis, and the treatment of other health issues related to the loss of endogenous estrogen production.