Background: Circulatingtumor cells (CTCs) are most commonly isolatedby using positive selection enrichment targeting the epithelial cell adhesion activating molecule (EpCAM). Using positive selection, a CTC is defined as EpCAM and cytokeratins (CK) positive (+) and CD45 negative (-). However, other rare cancer associated cells (RCACs) including EpCAM - and/or CD45 + cells have been reported (van de Stolpe, 2011). In order to investigate these RCACs, a prospective study in MBC using multi-parameter flow cytometry and immunocytochemistry (ICC) was undertaken. Methods:Forty blood samples from MBC pts were collected prior to initiation of a new line of chemotherapy, after one cycle, or at time of progression. Twenty one (52%) samples were from pts with estrogen receptor (ER) -, progesterone receptor (PR) -, and HER2neu non-overexpressing, or triple negative breast cancer (TNBC); nineteen (47%) were from pts with ER/PR +, HER2neu nonoverexpressing disease. Blood was processed using immunomagnetic negative depletion (Yang, 2009). Multiparameter, flow cytometry analysis was conducted prior to, and after depletion of CD45 + cells. Multistep, sequential labeling and fixation for surface markers was performed, followed by permeablizition for CK. ICC staining for EpCAM, CD45, CK 8/ 18/19 and DAPI was performed. Results: Either very low or no CK + events were present in blood samples from healthy volunteers (n=5). Various post-enrichment events were identified. These populations were also present by ICC with confocal microscopy. Conclusions: In addition to CTCs, RCACS that are CD45 + and/or EpCAM - are detectable in MBC pts and more frequently present in TNBC pts. These populations, only detectable by negative depletion, may be clinically significant as ongoing studies evaluate the predictive capabilities of blood based biomarkers.

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 2371. doi:1538-7445.AM2012-2371