Use of the DEPArray platform to detect, isolate, and molecularly characterize pure tumor cells from peripheral blood samples enriched using the CellSearch system.

Abstract

10616 Background: Circulating tumor cells (CTC) offer the potential for serially monitoring the molecular profile of a tumor. However, enrichment techniques provide a level of purity problematic for most molecular analysis methods, and do not readily provide for analysis of tumor cell heterogeneity. We evaluate the use of DEPArray (Silicon Biosystems), an automated system enabling image-based cell sorting with single-cell resolution, for CTC isolation and characterization from enriched blood samples. Methods: Experiments were carried out with healthy-donor blood (HB) collected in CellSave tubes, spiked with tumor cells (TC) and enriched on Veridex’s AutoPrep with the CellSearch Epithelial Cell Kit. DEPArray system was used for detection and multiple recoveries of single TCs (or control WBCs) and 5 cell batches. A comparison of blind enumeration results with Veridex’s CellTracks Analyzer II (CTAII) was carried out on replicate samples. Results: No TCs were detected among negative controls (n=10). TC count (normalized to sample volume analyzed) was compared sample-wise for each replicate (n=20): DEPArray/CTAII count was, on average, 100% (standard deviation = 52%). Enriched mixtures of Her2+ and Her2- TCs spiked in HB samples (n=5), were sorted by DEPArray and recovered into separate tubes. By phenotypical re-analysis no Her2+ cells were detected among the Her2- cell fraction and vice versa, neither were donor WBCs found (100% purity). KRAS-mutated, A549 cells spiked in HB were enriched and loaded on DEPArray. Individual fractions containing either 1 to 5 tumor cells or donor WBCs were sorted. Whole Genome Amplification (Ampli WGA, Silicon Biosystems), KRAS specific gene amplification and Capillary Electrophoresis sequencing were carried out. TCs successfully amplified showed only mutated KRAS, (WBCs were only wild-type). Conclusions: DEPArray achieved 100% purity, eliminating all white blood cells (WBC), in the isolation of a mixed population of tumor cell lines downstream of CellSearch enrichment. This enabled molecular profiling of pure tumor cells from whole blood spiked tumor cell lines.