ClearCell FX Microfluidic System for the Enrichment and Genetic Analysis of Circulating Tumor Cells.

Abstract

e22023 Background: Increasing evidence suggests that the enumeration and gene mutations associated with CTCs will provide a non-invasive means for individualizing cancer therapy, assessing prognosis, and treatment monitoring. ClearCell FX is a semiautomated microfluidics system that utilizes the biomechanical differences between tumor and non-tumor cells to isolate intact viable CTCs for cell enumeration and downstream molecular analyses. Methods: We evaluated the performance of ClearCell FX using spiked-in cancer cells H1299 and MCF7 in normal control blood. We also compared the performance of ClearCell FX to CellSearch using parallel blood samples from patients with advanced breast and colorectal cancers. Isolated CTCs from patients with early resectable lung cancer and metastatic colon and breast cancer are examined by immunofluorescence (IF) staining, fluorescence in situ hybridization (FISH), next generation sequencing (NGS) with the Ion AmpliSeq hotspot cancer panel, and whole exome sequencing. Results: ClearCell FX system provided up to 80% cell recovery after a single (1x) run. Recoveries were 60-70% after a second enrichment (2x) run. The rate of recovery was linear from 10-5000 cells and consistent for both lung and breast cancer cells (H1299 and MCF-7 cells, R2 = .988). Cell purity was less than 1% after 1x run but increased to 1-40% with the 2x protocol thus making it clinically feasible for downstream molecular analyses. The sensitivity to detect tumor specific mutations in CTCs by NGS in the ClearCell enriched sample was as low as 3 cells per ml of blood. NGS analyses of 2x enriched CTCs using clinical samples have so far detected missense variants in the SMO, CDH1 and MET genes. Conclusions: We validated the ClearCell FX system for robust, low-cost and label-free enrichment of CTCs. Our data shows that the cells recovered are suitable for IF and FISH and that the relatively high purity of the 2x run samples are readily usable for downstream NGS analyses. Disclosure Declaration: This work is supported by a collaborative research agreement with ClearBridge BioMedics.