511 Background: Trastuzumab is a humanized monoclonal antibody targeted to HER2 protein and currently indicated for HER2-positive breast cancer defined by overexpression of HER2 protein (3+ IHC staining by HercepTest™) or HER2 gene amplification (HER2/CEP17 ratio over 2 by PathVysion® FISH assay). These criteria were determined for advanced disease but have not been formally tested in the adjuvant setting. We examined these tests’ ability to predict benefit from adjuvant trastuzumab in NSABP trial B-31. Methods: All available tumor tissue blocks from the B-31 trial were subjected to HercepTest and PathVysion assay as defined in the B-31 protocol. Formal statistical test of interaction between HER2 levels measured by these two tests and benefit from trastuzumab was performed. Results: 207 of 1,795 cases (11.5%) showed gene amplification as determined by PathVysion, and 255 of 1,662 (15.3 %) showed overexpression as determined by HercepTest. 161 of 1,662 (9.7%) had neither gene amplification nor overexpress...