title: Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.
abstract: Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.
authors: Center of Predictive Molecular Medicine, University-Foundation, Chieti, Italy. amarchetti@unich.it
authors: Antonio
authors: A
authors: Marchetti
authors: Maela
authors: M
authors: Del Grammastro
authors: Giampaolo
authors: G
authors: Filice
authors: Lara
authors: L
authors: Felicioni
authors: Giulio
authors: G
authors: Rossi
authors: Paolo
authors: P
authors: Graziano
authors: Giuliana
authors: G
authors: Sartori
authors: Alvaro
authors: A
authors: Leone
authors: Sara
authors: S
authors: Malatesta
authors: Michele
authors: M
authors: Iacono
authors: Luigi
authors: L
authors: Guetti
authors: Patrizia
authors: P
authors: Viola
authors: Felice
authors: F
authors: Mucilli
authors: Franco
authors: F
authors: Cuccurullo
authors: Fiamma
authors: F
authors: Buttitta
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D017353:Gene Deletion; D018773:Genes, erbB-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008969:Molecular Sequence Data