Abstract P2-09-25: ERα Expression in Breast Cancer: A Conundrum of Antibody Specificity?
Authors: Wu X , Subramaniam M , Negron V , Lingle WL , Goetz MP , Ingle JN , Spelsberg TC , Hawse JR. 
Published: April 2011

Abstract
Background: The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity; however, much less is known about the role of ERß. In vitro studies demonstrate a tumor suppressive function for ERß, and we have recently implicated a role for ERα in sensitizing ERα expressing breast cancer cells to the anti-estrogenic effects of endoxifen. However, the in vivo relevance of ERα remains unclear due to conflicting reports. Here, we provide evidence that some of this controversy may be explained by variability in antibody specificity. In addition, we describe the development and characterization of a novel, highly specific monoclonal antibody and provide data regarding ERα expression in human breast cancers.
Methods: Five commercially available ERα antibodies were screened for their sensitivity and specificity using western blotting, immunoprecipitation, immunofluorescence and immunohistochemistry in known ERα negative and positive cell lines as well as in normal human tissue samples. A novel monoclonal ERα antibody (C10) was developed and characterized in the same manner. Following identification of two specific antibodies, ERα expression was assessed in 66 breast tumors collected prior to adjuvant therapy. Samples were scored separately for nuclear and cytoplasmic staining.
Results: In depth analysis of commercially available ERα antibodies reveled that the majority were non-specific with substantial cross-reactivity to ERα . Only one commercial antibody (PPG5/10), which solely recognizes full-length ERß, and our newly developed monoclonal antibody, which recognizes full-length and all 4 ERα variants, were determined to be sensitive and specific for ERα expression. These same two antibodies resulted in strong staining for endogenous levels of ERα protein in normal prostate tissue by immunohistochemistry. We further assessed these two antibodies in a set of breast tumors. Preliminary analysis revealed significant differences for ERα positivity between these two antibodies. Based on nuclear staining, 92% of tumors were ERα positive using the PPG5/10 antibody while only 34% were positive with C10. Approximately 50% of all tumors exhibited cytoplasmic staining with both antibodies. Conclusions: Our studies demonstrate that the majority of commercially available ERα antibodies are either non-specific or insensitive for the detection of ERα via immunohistochemistry. The present data call into question the relevance of prior studies which tested the association between clinical outcome and ERα expression and demonstrate the need to further analyze the role of ERα in breast cancer using highly specific and validated antibodies. While both the PPG5/10 and C10 antibodies are highly specific for ERß, the significant discrepancy in nuclear staining between them in breast tumors may be due to changes in epitope availability as a result of post-translational processing. Our newly developed C10 antibody could provide additional discriminatory features which may be useful in predicting response to therapy and/or associations with other clinicopathological factors and such studies are currently underway.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-25.