Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or initially not present. A prospective trial was designed to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM dependent enrichment/isolation technology.

A total of 32 patients with metastatic breast cancer were enrolled and blood samples were processed using previously described immunomagnetic negative depletion methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep, sequential labeling was performed to label and fix cell surface markers followed by permeablization for cytokeratin (CK) 8, 18, 19. Multiparameter flow cytometry analysis (FCM) was next conducted on a BD LSR, Aria II or Aria III. Immunocytochemical (ICC) staining on post-enrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor (EGFR) and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).

CD45 negative (-) CK positive (+) populations with EpCAM+ and EpCAM - expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM+ and EpCAM- populations that were CK+ and co-expressing the pan- hematopoietic marker CD45 were also noted. There were statistically significantly more CK+EpCAM- events/mL than CK+EpCAM+ events/mL in both the CD45- or CD45+ fractions (both p <= 0.0005). The number of CK+CD45- and CK+CD45+ events per mL of blood sample, (regardless of EpCAM status) were higher in patient samples than in normal control samples (p <= 0.0005, and 0.026, respectively). Further, a significant fraction of the CK+CD45+ events also expressed CD68, a marker associated with tumor associated macrophages (TAMs). Higher levels of CD45-CK+EpCAM- was associated with worse overall survival (p = 0.0292).

Metastatic breast cancer patients have atypical cells that are CK+EpCAM- circulating in their blood. Since a substantial number of these patients will not have EpCAM+CTCs, additional studies are needed to evaluate the role of EpCAM- circulating cells as a prognostic and predictive marker.