Characterization and identification of specific EGFR mutations in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients using antibody independent method, ApoStream.

Abstract

11044 Background: A variety of methods for isolation of CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM). Using classic phenotypic definition, a CTC is nucleated, cytokeratin CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStrea. For CTC enumeration comparison, CellSearch FDA-approved kit was used. Isolated cells were evaluated with multiplexed immunofluorescent assay and laser scanning cytometry analysis were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCam. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 32 NSCLC patients and 3 healthy volunteers were processed. ApoStream isolated 0 to 65 CK(+)/CD45(-) CTCs(n=32) and CellSearch isolated 0 to 13 EpCAM(+)/CK(+)/CD45(-) CTCs(n=7). Additionally, ApoStream™ recovered 37-3536 CK(-)/CD45(-) and 4-10702 CK(+)/CD45(+) cells. EpCAM expression was detected in 7-100% of CK(+)/CD45(-) and 0-5% of CK(-)/CD45(-) cells, and 18-100% of CK(+)/CD45(+) cells. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Complete mutation analysis will be presented.