Determination of Estrogen Receptors in Paraffin-Embedded Tissue: Techniques and the value in breast cancer treatment

Abstract

Estrogen receptor (ER) analysis in breast cancer has been used in three clinical situations: to select patients with advanced breast cancer for hormonal therapy, as a prognostic parameter, and for selection of women with early breast cancer to adjuvant hormonal treatment. ER has traditionally been measured using labelled hormone in binding assays—often in dextran-coated charcoal assays (DCC). Monoclonal antibodies to ER has permitted development of a solid phase enzyme immunoassay (ER-EIA) used for quantitative determination of ER in tissue homogenates, and have also been used for determination of ER using an immunohistochemical assay in frozen sections (ER-ICA) or in formalin-fixed, paraffin-embedded tissue (ER-PAR). A large number of studies has compared ER-EIA with ER-DCC assays. There is a good linear correlation between the two types of assay but ER-EIA measure more ER and classify a larger fraction of tumors ER-positive than conventional ER assays. Lack of clinical data makes the significance of this uncertain. Numerous studies have reported on the correlation between ER-ICA and ER-DCC or ER-EIA. There is a good correlation among the assays on classification of ER status with a median 86% concordance, but a somewhat poorer correlation between semiquantified ER of immunohistochemical assays and ER determined by the quantitative methods (median coefficient of correlation 0.67). There is a large variation in the cut-off level for definition of ER-positive in immunohistochemical assays emphasizing the need for quality control studies. The major problem involved in ER analysis in paraffin-embedded tissue is a considerable loss of immunoreactivity compared to sections from frozen tissue. This can partly be overcome by modifications of the immunohistochemical technique using enzyme pretreatment and other amplification systems, but the sensitivity of ER-PAR remains lower than ER-ICA despite these modifications, and the ER status is less reliably determined in tumors with low ER contents (< 100 fmol). The prognostic value of ER-PAR was evaluated with a multivariate analysis. The endpoint was disease-free interval in systemically untreated patients with early breast cancer, and the variables used were: ER-DCC, ER-PAR, age, tumor size, tumor grade, and nodal status. A total of 133 patients from the Danish Breast Cancer Cooperative Group's (DBCG) 77c protocols had a complete set of variables. The analysis showed that only nodal status, ER-DCC, and tumor grade were significant and independent prognostic variables. An overview of larger multivariate studies on mainly node-negative patients failed to show independent prognostic significance of ER-DCC. The predictive value of ER-PAR in adjuvant hormonal treatment of women with early breast cancer was examined in 349 patients from the DBCG 77c protocols randomized to radiotherapy or radiotherapy + tamoxifen. A previously published analysis at 42 months' follow-up had shown only women with > = 100 fmol ER/mg protein to benefit from tamoxifen. ER-PAR was determined in these patients and data reanalyzed. This showed tamoxifen to be without benefit in any of the receptor-defined subgroups at 86 months and that there was no difference between ER-PAR and ER-DCC. It is difficult on the basis of publicized studies, to interpret the relationship between ER and the benefit of adjuvant hormonal treatment, and most large studies fail to disclose receptor-defined subgroups who do not benefit from hormonal treatment. Stratification of patients using receptor status should thus be planned with caution. ER-PAR was analyzed in 137 previously untreated patients with advanced breast cancer. Fifty percent of the ER-PAR positive patients responded to endocrine treatment compared to 10% of the ER-PAR negative. This is comparable to results from ER analyses in fresh tissue. Studies on response to hormonal treatment in breast cancer and immunohistochemical analyses are generally very small and difficult to compare. ER-PAR was used to compare ER status of the primary tumor and its metastases. ER status was concordant in the primary and 84 of 92 regional lymph node metastases and in 44 of 51 distant metastases. The discordance is readily explained by methodological limitations. The observations cannot explain the lack of response in 40-50% of patients with ER positive primary tumors.