Abstract 4227: Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE).

Abstract

Abstract Transgenomic, Inc. has developed a size-based analysis for the detection of all EGFR Exon 19 deletions (including complex mutation) greater than 6 base pairs (ACE™ Kit EGFR Exon 19). Currently there are over 70 known EGFR Exon 19 deletions in the region important for response of non-small cell lung cancer (NSCLC) to thymidine kinase inhibitors (TKIs). The majority of these deletions are rare, but the patients may benefit from TKI treatment if the deletion is present. In most current testing paradigms, a NSCLC patient with one of these rare mutations would be considered EGFR mutation negative and not receive TKI therapy. We have designed an easy, size-based PCR assay to detect any of the deletions present in any sample after a single PCR reaction. The assay is based on the separation of PCR fragments of different sizes using microcapillary electrophyoresis (MCE). Transgenomic's WAVE® MCE platform can discriminate the deletions from the wild-type sequence in EGFR Exon 19, however the exact nature of the mutation cannot be determined. If the exact nature of the mutation is required, the PCR fragment can be sequenced. To confirm that the platform can detect common and rare mutations, plasmids containing various EGFR Exon 19 deletions were synthesized, mixed with EGFR wild-type plasmids and analyzed on the WAVE® MCE platform. The Limit of detection for the plasmid mixing experiments is between 0.5 and 1% for all plasmid constructs tested. These plasmids include 6, 9, 15, 21, 24, and 25 bp deletions . As all samples tested have some wild-type sequence present, 6 base pair deletions cannot be resolved from the wild-type fragment. Several of the deletion constructs contain those not detected by any allele-specific kit, however patients with these deletions, as determined by Sanger sequence analysis, have benefitted from TKI therapy. In addition cell line DNA and DNA isolated from FFPE sections have been analyzed for the detection of EGFR Exon 19 deletions and similar limits of detection (0.5-1.0%) were seen. This simple, size-based analysis for EGFR Exon deletions will result in NSCLC patients with both common and rare EGFR Exon 19 deletions being able to receive the treatment option best suited for their molecular profile. Citation Format: Katherine A. Richardson, Grant Wu, Rui Lin, Yanggu Shi, Phil Eastlake, Benjamin Legendre. Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2013-4227