Abstract 689: Validation of antibodies to Estrogen Receptor ≤ and quantitative assessment of ERα1 and ERα5 expression in breast cancer

Abstract

Abstract INTRODUCTION: Estrogen receptors are members of the nuclear hormone receptor family that play an important role in breast carcinogenesis and response to endocrine therapy. Though the role of ERα in breast cancer has been studied extensively, little is known about the alternative isoform ERα. ERα has significant sequence homology to ERα but is located on a different chromosome and maintains both overlapping and unique functional attributes. Five variants resulting from alternative splicing of the C-terminal region of ERα exist. The relevance of ERα variants in breast cancer outcomes and response to therapy is difficult to assess because of conflicting results in the literature, likely due to variable methods used to assess ERα in patient tumors. METHODS: Antibodies against ERα variants (ERα1: ThermoScientific PPG5/10; ERα2/cx: Serotec Clone 57/3; ERα5: Serotec Clone 5/25) were validated for staining specificity by siRNA knockdown of ESR2 as well as staining reproducibility on FFPE tissue by quantitative immunofluorescence (QIF) using AQUA technology (HistoRx). QIF staining of validated antibodies was then assessed on two separate breast cancer cohorts. RESULTS: ERα1 and ERα5, but not ERα2/cx, antibodies were found to be sensitive, specific and reproducible, as shown by reduction in signal after siRNA knockdown in cell lines and reproducible QIF scores on a set of breast cancer control cases. ERα1 and ERα5 expression is significantly associated in both cohorts examined (R*2=.224, p<0.0001). However, ERα1 does not show significant association with patient outcome or response to endocrine therapy. In contrast, ERα5 is associated with worse recurrence free survival (log rank p=0.0500) and, though it shows no association with response to tamoxifen, it predicts response to chemotherapy (log rank p=0.0393). CONCLUSIONS: Validation of ERα antibodies reveals that not all reagents are specific for the expected isoforms. Assessment of breast cancer cohorts using validated reagents show that ERα1 is not associated with outcome while ERα5 is both prognostic and predictive. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 689. doi:1538-7445.AM2012-689