Abstract C18: Epithelial and mesenchymal phenotypic characterization and mutation detection in circulating tumor cells isolated from peripheral blood of non-small cell lung cancer patients with ApoStream® technology
Authors: Balasubramanian Priya , Gupta Vishal , Hasegawa David , Lam Carmen Y. , An Ran , Gunturu Swetha Pratyusha , Tran Hai T. , Heymach John V. , Davis Darren W. 
Published: May 2016

Abstract
Background: The presence of circulating tumor cells (CTCs) has been established as a prognostic indicator in a few metastatic cancers. Although technically challenging, isolation and characterization of CTCs at the protein or nucleic acid level has significant potential to enable the development of minimally invasive methods to detect molecular alterations for personalized cancer targeted therapies. The goal of this study was to demonstrate the ability to enrich phenotypically heterogeneous population of CTCs and detect EGFR and KRAS mutations in isolated CTCs obtained from peripheral blood of patients with Non-small cell lung cancer (NSCLC) using ApoStream®, an antibody-independent CTC capture device based on dielectrophoresis technology.
Methods: For condition optimization, blood samples from a small cohort of cancer patients (n = 8) were collected under an IRB approved protocol and processed with multiple ApoStream® operating frequencies (45, 55, 65 and 85 kHz). To evaluate clinical feasibility, blood samples (8 mL) from 35 additional NSCLC patients (with or without targetable “driver” mutations) and 10 normal, healthy donor controls were collected and processed using the optimized ApoStream® conditions. CTC enumeration was performed using a multiplex phenotyping panel targeting hematopoietic (CD45/CD15), epithelial (EpCAM/CK/E-Cadherin) and mesenchymal (Vimentin/N-cadherin/Twist-1) markers. In addition, mutation detection assays were validated for EGFR (E746_A750, L858R, and T790M) and KRAS (G12C, and G12S) with mutant DNA spiked into wild type DNA at multiple dilutions using the droplet-based RainDrop® digital PCR system.
Results: An ApoStream® operating frequency of 85 kHz resulted in higher CTC counts and hence was selected for processing samples for CTC enumeration assay and a lower operating frequency of 65 kHz was selected to provide higher purity for processing samples for mutation analysis. Preliminary data demonstrated detectable CTCs (above healthy control baseline of 4 CTCs per mL blood) in 7 out of 10 patient samples processed to date (mean = 24; median = 14 and range = 5-100 CTCs per mL blood). CTCs isolated from all 7 patients exhibited mesenchymal phenotype. In addition, two patients also showed CTCs of epithelial phenotype demonstrating that the ApoStream® platform enriches phenotypically heterogeneous CTCs. Sensitivity of mutation detection determined from dilution series was found to be assay dependent; limit of quantitation (LOQ) for EGFR and KRAS mutations were found to be 0.01% or higher while the linearity for all the assays demonstrated a linear regression coefficient (R2) greater than 0.95 except for EGFR L858R which requires further analysis. Preliminary data from 8 NSCLC patient samples demonstrated the ability to detect mutations in ApoStream® enriched CTCs. Enumeration and mutation analysis is in progress for the remaining patient samples.
Conclusion: We demonstrated the ability to isolate, enumerate and define epithelial and mesenchymal phenotypes of CTCs and to detect oncogenic mutations in CTCs isolated from NSCLC patient samples using ApoStream®.
Funded by National Cancer Institute under Contract No. HHSN261201300070C.
Citation Format: Priya Balasubramanian, Vishal Gupta, David Hasegawa, Carmen Y. Lam, Ran An, Swetha Pratyusha Gunturu, Hai T. Tran, John V. Heymach, Darren W. Davis. Epithelial and mesenchymal phenotypic characterization and mutation detection in circulating tumor cells isolated from peripheral blood of non-small cell lung cancer patients with ApoStream® technology. [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 C18.