Potential impact of different methodologies for detecting EGFR mutations in selecting chemotherapy for patients with lung cancer.

Abstract

e19006 Background: Mutation analysis of the kinase domain of EGFR (exons 18-21) is a standard recommended procedure for patients diagnosed with non-small cell lung cancer (NSCLC). NSCLC patients whose tumor harbors certain EGFR mutations have notable responses to EGFR inhibitors. Several different methodologies are available as published assays or commercially available kits and include Sanger Sequencing, allele specific PCR (ASP) and restriction fragment length polymorphism (RFLP). Differences in the design of these three assays dictate which clinically relevant mutations will be detected. Methods: Sanger Sequencing of EGFR found 518 potentially clinically actionable mutations and 45 variants of unknown significance of the 4307 samples tested. To assess which clinically relevant EGFR mutations will be detected by the ASP and RFLP, we performed an in silico analysis of all observed mutations against the design specification of the ASP and RFLP assays. The RFLP assay used in this assessment was developed in our laboratory and is designed to detect all G719 mutations, all exon 19 deletions, all exon 20 insertions and the specific mutations T790M, L858R, L861R and L861Q. A commercially available ASP kit designed to detect 29 mutations in the kinase domain was also analyzed in this study. Results: Based on the performance characteristics assumed by the RFLP and ASP assays, the analytical sensitivities for detecting clinically actionable mutations of the two methodologies are 98.8% and 86.7% respectively, when compared to Sanger Sequencing. It is notable that the RFLP assay missed the S768I mutation (0% detected) whereas the assay did not detect some G719 mutations, exon 19 deletions, and exon 20 insertions (76.3, 86.6 and 35.3% detected respectively). Conclusions: Sequencing is still the preferred method of mutation detection in EGFR for NSCLC as it is the most comprehensive. However, if tumor nuclei are limited a more sensitive method than sequencing is required; EGFR mutation detection by RFLP would identify more of the potentially clinically relevant mutations than ASP. The ASP method would produce false negative results in 13.3% of patients expected to respond to EGFR inhibitors.