136 Background: Since only 40% of HER2-positive breast cancer patients respond to trastuzumab therapy, there is a great clinical need for a test that will better predict which patients would be responders to trastuzumab therapy. Since no single biomarker will likely improve identification of responders, this study will determine the efficacy of multiple biomarkers to predict response of patients to trastuzumab therapy. Our company has developed a protein analysis platform, layered immunohistochemistry (L-IHC), for the analysis of multiple protein biomarkers from a single FFPE tissue section while retaining the histologic orientation. L-IHC is being used to develop a companion diagnostic test in which multiple protein biomarkers are analyzed in HER2-positive breast cancer specimens to predict which patients will respond to trastuzumab therapy. Methods: In this ongoing clinical exploratory study, analysis of pretreatment archival HER2-positive FFPE breast cancer tissue sections from patients whose subsequent therapy included trastuzumab and whose response to therapy (responders and non-responders) is known was performed using L-IHC. Tissue sections were probed using a panel of 14 biomarkers along the HER2 and mTOR pathways were analyzed. In a blinded fashion to the response data, the intensity of specific biomarker signals corresponding to cancer regions on an H&E was visually scored (0,1,2,3, & 4+). The pattern of biomarker expression was correlated with the responder status of the patient. Results: A total of 24 responders and 9 non-responders were analyzed. The sum of four of the 14 biomarkers including p-mTOR, p-4EBP1, pERK, and HIF1-alpha discriminated the responders and non-responders. Using 7 as a cutoff, this test correctly identified 21/24 responders and 7/9 non-responders for a sensitivity of 87%, specificity of 78%, and an accuracy of 82%. Conclusions: Results of this study strongly suggests that analysis of p-mTOR, p-4EBP1, pERK, and HIF1-alpha using L-IHC improves twofold the prediction of patient response to trastuzumab compared to Her2 alone.