Abstract 3029: Profiling HER3/ErbB3 activation in formalin-fixed, paraffin-embedded (FFPE) breast tumor samples that express high and low HER2/ErbB2 levels using proximity-based immunoassays.

Background: HER3 is widely accepted as a potent heterodimerization partner for HER2 tyrosine kinase. HER3 activation may promote intrinsic and/or acquired resistance to HER2-targeted therapies. Therapeutic strategies that target HER3 directly or in combination with HER2 are being evaluated both preclinically and clinically. Historically, such strategies have focused exclusively on tumors that exhibit HER2 gene-amplification and/or over-expression (HER2 high/positive). Recent studies suggest distinct or additional utility in non-amplified, low HER2 tumor settings (HER2 low/negative). Attempts to identify appropriate HER2-positive patient populations that are likely to benefit from HER3-targeted therapies have revealed an important need for more quantitative, sensitive methods of measuring HER2 and HER3 protein expression other than traditional IHC, and also for quantitative, directed measurements of HER3 activation. Methods: Dual-antibody proximity-binding assays (VeraTag) were used to measure HER2 total (HERmark®), HER3 total, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase (p85) protein expression across a broad dynamic range in a group of HER2-(low) and HER2+ (high) FFPE breast tumors (HER2 status determined by IHC and verified by HERmark). Dual-antibody proximity-binding assays developed for use in high HER2 FFPE tumor specimens (PloS ONE: 28 Jan 2011 10.1371/journal.pone.0016443) were optimized for use in low HER2 samples. Results: HER2 expression spanned nearly a 2-log10 dynamic range in the HER2 breast tumor set. HER3 expression spanned a 1.5-log10 dynamic range in these tumors and trended with HER2 expression levels. HER2-HER3 heterodimerization correlated more closely with HER2 expression than HER3 expression in both low and high HER2 breast tumors, suggesting that HER2 may be the primary driver of heterodimerization. Correlations between phospho-HER3, HER3-PI3K, and HER2-HER3 heterodimer measurements were observed, with each analyte spanning a ∼1-log10 dynamic range for the corresponding measurement. HER3 signaling patterns in low and high HER2 breast tumors were compared to assess the level of HER3 activation relative to HER2 expression levels. Conclusions: We have developed multiple assays for quantitative measurements of HER3 activation in FFPE breast tumors samples that express low and high levels of the HER2 receptor. These assays are presently being investigated preclinically to define mechanism of drug action, and in clinical trials to predict response to targeted therapies. Citation Format: Jerry Wallweber, Ahmed Chenna, Roy Ravanera, Weidong Huang, David Stathas, Gayle Marshall, Chris Womack, Alison Pritchard, Ken Thress, Carl Barrett, Christos Petropoulos, Lisa DeFazio Eli, John Winslow. Profiling HER3/ErbB3 activation in formalin-fixed, paraffin-embedded (FFPE) breast tumor samples that express high and low HER2/ErbB2 levels using proximity-based immunoassays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2013-3029