Identification of actionable mutations in patients' tumors is essential in guiding therapy. The aims of this study were: a) to validate mutation detection by next-generation sequencing (NGS) in a cohort of NSCLC patients and b) to identify molecular subgroups within EGFR wild-type and KRAS mutant NSCLCs
We used the Ion Torrent AmpliSeq Colon and Lung cancer panel to analyze formalin-fixed paraffin-embedded tumors from 76 NSCLC patients previously tested for EGFR mutations by Sanger Sequencing. The sensitivity of the method was assessed by using commercial reference FFPE standards with defined allelic frequencies. DNA was isolated from microdissected tumor tissue and sequencing was performed in the Ion PGM platform.
Tumors were sequenced to a median coverage of 650X. The sensitivity of mutation detection was estimated at 4% and for mutation reporting we have used a baseline prevalence of ≥5%. Precision of the method was demonstrated by analyzing four tumor specimens two times in different library preparations and runs. A complete concordance was observed between the previously defined Sanger genotypes and the corresponding variants detected using NGS. A single mutation was detected in 30 of 76 (39.5%) specimens, two in an additional 30 (39.5%) whereas mutations in more than two genes were detected in 11 (14.5%). The most frequently mutated genes were TP53 (41/76; 54%) and KRAS (23/76; 30%). Among KRAS mutated tumors, 3 (13%) carried STK11, 2 (8.7%) kinase inactivating BRAF mutations and one (4.4%) the BRAF V600E. Between the EGFR/KRAS wild type tumors, 5 (13%) had a mutation in the PI3K pathway, 3 (7.3%) carried mutations in MET, 2 (4.9%) had the BRAF V600E and 6 (14.6%) carried STK11 loss of function mutations
NGS can be used for molecular diagnostics in NSCLC and may detect additional mutated pathways that can be targeted using novel therapies
Clinical trial identification
Legal entity responsible for the study
Medical School, University of Crete
University of Crete
All authors have declared no conflicts of interest.