e11132 Background: Changes in the tumor biomarker status between primary site and metastatic lesions are known to occur at a significant frequency. Correlating biomarker status in primary and metastatic tumors as well as circulating tumor cells (CTCs) may provide critical information for overall diseases pathogenesis. Here we report correlative profiling of tumor cells obtained from metastatic regions and CTCs to primary disease. Methods: The Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. In this study, fine needle aspirate (FNA) samples were collected from various metastatic sites (lymph nodes, lung, liver, chest well, etc.) from 58 breast cancer (BCA) patients and matching CTCs for correlative analysis of HER1, HER2, p95HER2, HER3, cMET, cKIT, IGF-1R, PI3K, AKT, ERK and other pathway proteins for their expression/activation. Results: Using the CEER assay, a comprehensive profile of pathway proteins can be determined from limited amounts of cancer cells in FNAs taken from metastatic sites. HER2 expression and activation was shown to be the primary pathway in some patients; however, a significant subset of HER2 positive patients also showed expression/activation of other redundant pathway proteins such as HER3, cMET, and IGF1R as well as truncated HER2 (p95HER2). Expression/activation of other targetable pathway proteins were found in BCA-FNAs with no HER2 activity. Varying levels of HER2 expression/activation were found in CTCs. The concordance of HER2 status between primary tumor, metastatic-FNAs and CTCs will be presented when samples IDs are un-blinded. Conclusions: The sensitivity and specificity of CEER assay is sufficient to provide a single-cell-level target phosphorylation. This enables profiling of pathway proteins in samples with limited amount of tumor cells, allowing frequent monitoring of tumor profile during the course of the treatments. Determination of comprehensive pathway analysis is critical in selecting the most effective therapy options and overcoming potential drug resistance through treatment combination or sequencing.