e21148 Background: Enumeration of circulating tumor cells (CTCs) in peripheral blood represents a prognostic marker for select solid tumors. Commercially available technologies for CTC detection rely on enrichment of EpCAM-expressing cells. However, not all tumor histologies demonstrate high constitutive EpCAM expression, including renal cell carcinoma (RCC). New techniques for more complex immunophenotypic characterization of CTCs are therefore of great interest. Methods: The AccuCyte system for characterization of rare blood cell populations retains all nucleated cells for analysis. Fluorochrome-conjugated monoclonal antibodies (mAbs) are added to 3 ml peripheral blood samples processed by density separation with a specialized tube and float. Centrifugation deposits all nucleated cells (Buffy coat) onto a surface designed for imaging with an automated fluorescence microscope. Sensitivity for CTC identification was measured by spiking blood from healthy donors with defined numbers of MDA-MD-453 breast tumor cells analyzed with mAbs to EpCAM and CD45. Peripheral blood samples from 4 patients (pts) with metastatic RCC and whose primary tumor had been resected were analyzed with a mAb specific for the RCC-associated antigen carbonic anhydrase IX (CAIX), as well as mAbs for CD10, EGFR and EpCAM. Results: In 13 blood samples from healthy donors, an average of 89% of spiked-in MDA-MD-453 tumor cells defined as EpCAM+/CD45- were detected by AccuCyte analysis with linearity from 0 to 139 cells (slope=0.8899; r2=0.9351) and a sensitivity of approximately 1 CTC per 106 nucleated blood cells. 3 of 4 peripheral blood samples from pts with clear cell RCC contained putative CTCs (range 1-6 cells/ml) defined as CD45-/CAIX+ nucleated cells. 3 CTCs from one patient were also CD10+ or EGFR+. All putative CTCs were EpCAM-. Conclusions: The detection of EpCAM-/CAIX+ cells in RCC pts demonstrates the adaptability of the AccuCyte platform for CTC (or other rare blood cell) identification defined by customized markers. Further development of marker panels for CTC analyses in pts with RCC and other tumor histologies as well as automation of image analysis is ongoing.
Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells