BRAF , KRAS , and EGFR Mutations Using Next-Generation Sequencing as Compared with FDA-Cleared Kits

摘要

Abstract Introduction We compared mutations detected in EGFR , KRAS , and BRAF genes using next-generation sequencing (NGS) and confirmed by Sanger sequencing with mutations that could be detected by FDA-cleared testing kits. Methods Paraffin-embedded tissue from 822 patients was tested for mutations in EGFR , KRAS , and BRAF by NGS. Sanger sequencing of hot spots was used with locked nucleic acid to increase sensitivity for specific hot-spot mutations. This included 442 (54%) lung cancers, 168 (20%) colorectal cancers, 29 (4%) brain tumors, 33 (4%) melanomas, 14 (2%) thyroid cancers, and 16% others (pancreas, head and neck, and cancer of unknown origin). Results were compared with the approved list of detectable mutations in FDA kits for EGFR , KRAS , and BRAF . Results Of the 101 patients with EGFR abnormalities as detected by NGS, only 58 (57%) were detectable by cobas v2 and only 35 (35%) by therascreen. Therefore, 42 and 65%, respectively, more mutations were detected by NGS, including two patients with EGFR amplification. Of the 117 patients with BRAF mutation detected by NGS, 62 (53%) mutations were within codon 600, detectable by commercial kits, but 55 (47%) of the mutations were outside codon V600, detected by NGS only. Of the 321 patients with mutations in KRAS detected by NGS, 284 (88.5%) had mutations detectable by therascreen and 300 (93.5%) had mutations detectable by cobas. Therefore, 11.5 and 6.5% additional KRAS mutations were detected by NGS, respectively. Conclusion NGS provides significantly more comprehensive testing for mutations as compared with FDA-cleared kits currently available commercially.