Background: Current established methods of circulating tumor cell (CTC) isolation and identification rely on antibodies against epithelial specific markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). The classical phenotypic definition of a CTC is a CK positive, CD45 negative, nucleated cell, yet several reports have shown that EpCAM and CK detect only a fraction of CTCs and are not sufficient to detect the heterogeneous subpopulations of CTCs. Moreover, subsets of primary tumor cells acquire features of invasiveness and transform into an aggressive phenotype. During this process, EpCAM and CK are down regulated or lost leaving a lethal population of CTCs undetectable and unstudied using antibody dependent CTC technologies. It is imperative to isolate CTCs in an unbiased, EpCAM independent manner and expand the phenotypic characterization of CTCs to elucidate the subpopulation heterogeneity. Here we used ApoStream™, a novel, antibody-independent device which exploits differences in the dielectric properties between cancer cells and normal blood cells to enrich CTCs from the blood of cancer patients. We demonstrate device performance and integration with additional methods to perform subsequent phenotyping and molecular marker analysis. Methods: The performance of ApoStream™ was assessed using SKOV3 (ovarian cancer) and MDA-MB-231 (breast cancer) cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. A side-by-side comparison of CellSearch® and ApoStream™ was performed on 10 metastatic breast cancer patients. A multiplexed immunofluorescent assay and laser scanning cytometry (LSC) analyses were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI cells, expression of EpCAM and vimentin. Results: In system precision performance studies, the average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 million peripheral blood mononuclear cells obtained from 7.5 mL normal donor blood was 75.4 ± 3.1% (n = 12) and 71.2 ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation (CVs) of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4-2600 cells. ApoStream™ consistently recovered significantly higher numbers of CTCs compared to CellSearch® (p = 0.024). ApoStream™ recovered varying numbers of CK+/CD45/DAPI+, CK+/CD45+/DAPI+, CK/CD45/DAPI+ cells from each cancer patient sample tested. ApoStream™ recovered both EpCAM+ and EpCAM CTCs in 50% and 90% of patients, respectively. Vimentin+ CTCs were isolated from 90% of patients. Conclusions: The ApoStream™ technology circumvents dependence on expression of EpCAM and recovers CTCs in high percentage of patients. ApoStream™ coupled with LSC analysis is a sensitive method for phenotyping and detecting biomarker expression in CTCs. These results demonstrate the broad applicability of ApoStream™ for enrichment and molecular characterization of CTCs as a foundation for improved clinical applications of CTCs.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-05.