Background: Profiling signaling pathway activation in cancer is critical as these pathways represent targets for new molecular guided therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides a unique series of biopsy specimens that have been subjected to extensive and detailed molecular characterization, together with detailed clinical and radiographic information. Methods: Purified tumor epithelium from 149 frozen pre-treatment biopsy specimens were procured via laser capture microdissection. Reverse phase protein microarray (RPMA) technology was used to quantitatively measure the activation of 40 key signaling proteins including components of pathways of interest for targeted therapy in breast cancer (HER2, IGFR, EGFR, mTOR, AKT and PLK1). Association between pathway activation and genomic subtype, pCR, residual cancer burden (RCB), and other clinical measures including tumor size, and nodal involvement was...