Author(s): Raffaella Pasquale aff1 , Francesca Fenizia aff1 , Riziero Esposito Abate aff1 , Alessandra Sacco aff1 , Claudia Esposito aff1 , Laura Forgione aff1 , Anna Maria Rachiglio aff1 , Simona Bevilacqua aff2 , Agnese Montanino aff3 , Renato Franco aff4 , Gaetano Rocco aff5 , Gerardo Botti aff4 , Marc G Denis aff6 , Alessandro Morabito aff3 , Antonella De Luca aff2 , Nicola Normanno [*] aff1 aff2
cftDNA; EGFR mutations; liquid biopsy; non-small-cell lung carcinoma; PNA-clamp; Therascreen®
Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of lung carcinoma cases, and is classified into three histological types: adenocarcinoma, squamous cell carcinoma and large cell carcinoma [1 ]. Genomic alterations affecting actionable signaling pathways have been identified in up to 75% of lung adenocarcinomas [2,3 ]. In particular, activating mutations of the EGFR have been discovered following analysis of the EGFR gene in patients that responded to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib in early clinical trials [4,5 ]. Although more than 250 mutations of the EGFR have been described up to now, two mutations, a single point mutation in exon 21, the p.L858R and a series of small in-frame deletions in exon 19, account for approximately 90% of all EGFR mutations [4 ]. Both mutations result in activation of the tyrosine kinase domain and have been associated with sensitivity to EGFR TKIs [6 ]. Several clinical trials demonstrated that in NSCLC patients carrying mutations of the EGFR , first-line treatment with EGFR TKIs is associated with a higher response rate and a longer progression-free survival as compared with chemotherapy [ 7-9 ]. Because gefitinib, erlotinib and afatinib have been approved in Europe for treatment of EGFR mutant NSCLC, international and national guidelines recommend EGFR testing for patients with lung cancer in order to choose the most appropriate first-line treatment [10,11 ].
EGFR testing is usually performed on tissue, but a significant fraction of patients do not have sufficient amount of material for mutational analysis. In this respect, analysis of liquid biopsy, in other words, circulating tumor cells or circulating free tumor DNA (cftDNA), is emerging as a new approach for tumor genotyping [12,13 ]. CftDNA is released into the bloodstream by tumor cells, with mechanisms that have not been completely elucidated yet. Most likely it results from tumor necrosis, from lysis of circulating tumor cells or from active release [14 ]. Due to its stability and ease of isolation, cftDNA could be a surrogate or an additional source of genetic material, and could represent a valid noninvasive alternative to biopsy for the molecular characterization of the tumor and to follow the mutational evolution of the disease over time [ 12,14,15 ]. Several different studies have suggested that EGFR mutations can be detected in patients with metastatic NSCLC by analyzing the cftDNA isolated from plasma or serum. In particular, both the Therascreen ® EGFR RGQ Mutation Kit, and a PNA-clamp approach have been shown to have a 60-65% sensitivity in detecting EGFR mutations in liquid biopsies [16-19 ]. The Therascreen kit is based on Amplification Refractory Mutation System allele-specific PCR and Scorpions probes, whereas...