Limits and potential of targeted sequencing analysis of liquid biopsy in patients with lung and colon carcinoma

Anna Maria Rachiglio, Riziero Esposito Abate, Alessandra Sacco, Raffaella Pasquale, Francesca Fenizia, Matilde Lambiase, Alessandro Morabito, Agnese Montanino, Gaetano Rocco, Carmen Romano, Anna Nappi, Rosario Vincenzo Iaffaioli, Fabiana Tatangelo, Gerardo Botti, Fortunato Ciardiello, Monica R. Maiello, Antonella De Luca, Nicola Normanno

The circulating free tumor DNA (ctDNA) represents an alternative, minimally invasive source of tumor DNA for molecular profiling. Targeted sequencing with next generation sequencing (NGS) can assess hundred mutations starting from a low DNA input. We performed NGS analysis of ctDNA from 44 patients with metastatic non-small-cell lung carcinoma (NSCLC) and 35 patients with metastatic colorectal carcinoma (CRC). NGS detected EGFR mutations in 17/22 plasma samples from EGFR-mutant NSCLC patients (sensitivity 77.3%). The concordance rate between tissue and plasma in NSCLC was much lower for other mutations such as KRAS that, based on the allelic frequency and the fraction of neoplastic cells, were likely to be sub-clonal. NGS also identified EGFR mutations in plasma samples from two patients with EGFR wild type tumor tissue. Both mutations were confirmed by droplet digital PCR (ddPCR) in both plasma and tissue samples. In CRC, the sensitivity of the NGS plasma analysis for RAS mutations was 100% (6/6) in patients that had not resection of the primary tumor before blood drawing, and 46.2% (6/13) in patients with primary tumor resected before enrollment. Our study showed that NGS is a suitable method for plasma testing. However, its clinical sensitivity is significantly affected by the presence of the primary tumor and by the heterogeneity of driver mutations.