Abstract 1707: A real-time PCR assay for detecting EGFR mutations in formalin-fixed paraffin-embedded tissue (FFPET) specimens of non-small cell lung cancer (NSCLC)

EGFR mutations identify patients with NSCLC who have a high likelihood of benefiting from first-line treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be time-consuming, and has limited sensitivity for low levels of mutations. We describe the analytic performance of the cobas EGFR Mutation Test, a multiplex, 3-reaction real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The mutations detected and the mutation report calls are listed below: The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5-micron FFPET section, and the test can be performed in 95% hit rate was obtained in blends with >5% mutant alleles for L858R and exon 19 deletions as determined by 454 sequencing (quantitative massively parallel pyrosequencing) at a total DNA input of 150ng, or 50 ng per PCR amplification. The cobas test was compared to 2x bidirectional Sanger sequencing using a set of 152 NSCLC FFPET specimens. The overall percent agreement (OPA) between the 2 methods was 96.7% (negative agreement - NPA 97.5%; positive agreement - PPA 95.8%). Specimens with discordant cobas EGFR Test and Sanger results were analyzed by 454 and a revised agreement analysis was performed based on the composite results of the 3 assays. The revised OPA was 98.7% (NPA 98.8%; PPA 98.6%). The call repeatability of a panel of NSCLC FFPET specimens was 98% when tested with two operators, instruments, and reagent lots. Necrotic tissue, hemoglobin, triglycerides, and common respiratory organisms did not interfere with the assay. The test showed no cross reactivity with the corresponding exon sequences from the HER2, HER3, and HER4 genes. These analytic studies demonstrate that the cobas EGFR Mutation Test is a sensitive, accurate and reproducible assay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1707. doi:1538-7445.AM2012-1707