EF-FACS circulating tumor cell detection complements ctDNA-based prognosis in stratification of non-small cell lung cancer patients

Abstract Purpose Liquid biopsy provides a real-time dynamic evolution of Non-Small Cell Lung Cancer (NSCLC). Circulating Tumor Cells (CTC) capture tumor heterogeneity enabling single-cell approaches, but their detection requires an enrichment process associated with cell loss. We present an Enrichment-Free Fluorescence Activating Cell Sorting method (EF-FACS), which permits the detection of a higher proportion of CTCs, and aimed to explore the complementary information of EF-FACS CTCs and ctDNA. Methods EF-FACS is based on fixation, permeabilization and immunostaining (CD45, CD66b, CK, EpCAM, Hoechst) of 12 mL whole blood, to detect and isolate CTCs by FACS. Tumor origin of CTCs is evaluated with a Low-Pass Whole-Genome copy number altered profile. Accuracy was assessed with tumor cells spiked in healthy blood samples. Blood samples of 95 advanced NSCLC patients were collected. 86 paired samples underwent CellSearch and EF-FACS workflows. 85 patients with a ctDNA analysis were included for combined CTC-ctDNA clinical utility. Results EF-FACS was accurate (r = 0.937, p = 1.689e-11). CTC counts were significantly higher by EF-FACS than CellSearch (median CTC counts: 6 vs 0, p < 0.0001). CTCs and ctDNA were found to be independent prognostic biomarkers. CTCs improved the short-term prediction of clinical prognosis factors (p = 0.0212), whereas ctDNA did not (p = 0.537). The combination of CTCs and ctDNA was complementary for enhanced stratification of the risk of death at 1 year in patients with advanced NSCLC. Conclusions EF-FACS is efficient in NSCLC CTC detection, potentially implementable in treating centers using a cytometer. CTCs and ctDNA have a complementary role in prognosis stratification of metastatic NSCLC patients.