Abstract C16: The use of an antibody independent method, ApoStream™, to isolate circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients and identification of EGFR mutations
Authors: Tran Hai T. , Anne Tsao S. , Richardson Katherine , Legendre Ben , Haider Asifa , Davis Darren , Heymach John 
Published: May 2016

Abstract
Background: A variety of methods for capture of rare CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). Using a classic phenotypic definition, a CTC is a nucleated, CK (+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells that 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 ApoStream™. For CTC enumeration comparison, the CellSearch® FDA-approved kit was used. Isolated cells were evaluated with a multiplexed immunofluorescent assay and laser scanning cytometry was 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 40 NSCLC patients and 12 healthy volunteers were processed. In the normal, healthy volunteers, ApoStream™ isolated 0-1 CK(+)/CD45(-) cells and 0-33 CK(+)/CD45(+) cells. From the 38 of 40 NSCLC patients, ApoStream™ identified 0 to 65 CK(+)/CD45(-) CTCs, 2 samples failed in processing. 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. In comparison, CellSearch® isolated 0 to 13 EpCAM(+)/CK(+)/CD45(-) CTCs in 7 patient samples tested. From our whole-blood spiked cancer cell (H1600, H1975) experiments, CTC recovery ranged from 13% to 60% with detection of EGFR mutations in as low as 10 recovered cells by ICE COLD PCR. From the 35 patients where CTCs were isolated by ApoStream, ICE COLD PCR correctly identified mutation status in 12 cases with EGFR exon 19 deletions (5), exon 21 - L858R(2) and wild type in 5 cases. There were 6 cases with either exon 18 or 20 mutation from tissue anlaysis that tested negative for exon 19 and 21 by ICE COLD PCR; exon 18 and 20 were not tested at this time. Mutation status was not detected in 16 cases when compared to tumor tissue analysis by Sanger sequencing. In 1 case, tissue revealed exon 19 - 15bp deletion meanwhile, CTC-ICP detected mutation at exon 21. Mutations were not tested in 5 CTC samples.
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 a subset of samples, higher number of CK(+)/CD45(-) cells were recovered by ApoStream™ than CellSearch®. Furthermore, from recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and utility of CTCs as an alternative to tissue biopsy.
Citation Format: Hai T. Tran, Tsao S. Anne, Katherine Richardson, Ben Legendre, Asifa Haider, Darren Davis, John Heymach. The use of an antibody independent method, ApoStream™, to isolate circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients and identification of EGFR mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C16.