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 (<5ng), an average depth of 6897 reads and 97% coverage was achieved in NGS analysis. Expected mutations were detectable in samples down to 10 spiked tumor cells (0.5% CTC purity). Analysis of CTCs from clinical samples is currently on-going.
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.