Abstract Survival rates of metastatic breast cancers (mBCAs) are considerably low. Often, tumor cells at the primary site may not reflect the profile of the tumor cell population in recurrent disease. Circulating tumor cells (CTCs) isolated from peripheral blood offer a non-invasive disease monitoring modality. Identification of reliable molecular markers within (CTCs) from patients with recurrent disease may further improve breast cancer survival. Hence, we have developed a multiplexed immunoassay for monitoring activated HER2 protein and their expression levels using a multiplexed immuno-microarray platform. Collaborative Enzyme Enhanced Reactive-immunoassay (CEER™) was utilized to analyze HER2 profiles in CTCs isolated from 76 BCA patients (stages III to IV) with HER2 negative primary disease. The CEER technology utilizes the formation of a unique immuno-complex requiring co-localization of two detector antibodies around capture antibodies immobilized on immuno-array to profile HER2 protein expression and activation. The collaboration between two channeling-enzymes conjugated on two detection antibodies in proximity, enables the profiling of the target proteins with extreme sensitivity and specificity. Approximately 25% of HER2 negative BCA patients in this cohort showed varying levels of HER2 activation in CTCs isolated from the recurrent disease. About 8% of patients with HER2 activation in CTCs also showed significant HER2 over-expression. This suggests mechanisms of HER2 activation either through formation of receptor heterodimer (in samples without HER2 over-expression) or homodimer (in samples with HER2 overexpression) formation. The distinct HER2 discordance between primary tumor and recurrent disease demonstrates an urgent need for routine monitoring of HER2 status in CTCs found in mBCA patients. Incidence of HER2 alterations in CTCs should be considered in selecting effective treatment regimens for BCA patients with relapsed disease. Furthermore, CEER can be used for profiling other druggable targets for guiding effective clinical strategies including rational targeted agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1217. doi:1538-7445.AM2012-1217