Routine Clinical Mutation Profiling of Non–Small Cell Lung Cancer Using Next-Generation Sequencing

Abstract

Context The availability of massive, parallel-sequencing technologies makes possible efficient, simultaneous detection of driver and druggable mutations in cancer. Objective To develop an amplicon-based, next-generation sequencing, mutation-detection assay for lung cancer using the 454 GS Junior (Roche Applied Science, Indianapolis, Indiana) platform. Design Fusion primers incorporating target sequence, 454 adaptors, and multiplex identifiers were designed to generate 35 amplicons (median length 246 base pairs) covering 8.9 kilobases of mutational hotspots in AKT1, BRAF, EGFR, ERBB2, HRAS, KRAS, NRAS, PIK3CA, and MAP2K1 genes and all exons of the PTEN gene. Results The assay was validated on 23 formalin-fixed, paraffin-embedded lung cancer specimens. A minimum number of reads was consistently achieved with overall median read depth of 529× per amplicon. In total, 25 point mutations and 4 insertions/deletions (indels) with a frequency of 5.5% to 93.1% mutant alleles were detected. All EGFR, ERBB2, KRAS, PIK3CA, KRAS, and PTEN mutations, as detected by next-generation sequencing, were confirmed by pyrosequencing, with the exception of 3 point mutations in a tumor sample with low mutant-allele burden (below the pyrosequencing limit of detection). Conclusions The GS Junior–based, targeted, resequencing assay for a focused set of non–small cell lung cancer driver genes allows for quick and sensitive detection of point mutations and indels for the most relevant therapeutic genes in this type of cancer.