High Purity Isolation Of Circulating Tumor Cells For Next-Generation Sequencing

Background: Being able to capture circulating tumor cells (CTCs) and characterize them at a molecular level promises novel insights into the mechanisms that drive metastasis, tumor recurrence and drug resistance. However, next generation sequencing (NGS) of CTCs has been challenged by antibody-enrichment bias, low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we describe the results of a new label-free protocol for CTC enrichment and NGS analysis. Methods: The lung adenocarcinoma cell line, NCI-H1975, with EGFR T790M and L858R mutations, and colorectal cancer cell line HCT-116, with KRAS G13D mutation, were fluorescently labelled and spiked in concentrations of 250, 50, 10 and 0 cells per 7.5 mL healthy volunteer blood in duplicate. CTCs were enriched using a modified protocol of the label-free spiral microfluidics-based ClearCell® FX system. DNA was extracted by QIAamp Micro kit and sequenced using the Ion AmpliSeq Cancer Hotspot Panel and Ion Torrent PGM system. Results: Imaging analysis indicated that CTCs were efficiently enriched (40-70% recovery) with extremely low leukocyte background (650-1300 cells). Despite a low DNA quantity ( Conclusion: The new protocol generated sufficient label-free CTC purity for NGS analysis, overcoming current limitations in the field, and facilitating discovery and clinical application. Citation Format: Nur Lina Mohd Salleh, Yi Fang Lee, Mei Hui Tan, Michelle A. Rosario, Ross A. Soo, Ali Asgar Bhagat, Richie Chuan Teck Soong. High purity isolation of circulating tumor cells for next-generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1603. doi:10.1158/1538-7445.AM2015-1603