Abstract 1390: High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR
Authors: Wu Grant , Jensen Sheena , Scott Karissa , Montagne Erin , Stoddard Jason , Krzycki Phil , Schweikart Courtney , Legendre Benjamin L. , Eastlake Philip , Richardson Katherine A. 
Published: July 2016

Abstract
Introduction: The EGFR Exon 20 T790M mutation confers resistance to the first generation tyrosine kinase inhibitors (TKI), such as Tarceva®. A new generation of treatments, including AZD9291, targets the T790M mutation thus allowing a new approach to cancer therapy for NSCLC patients. Recently EGFR Exon 20 C797S mutations (c.2389T&gt;A and c.2390G&gt;C) have been identified as resistance mutations to the third generation TKI AZD9291. High sensitivity detection of these mutations, as well as the T790M mutation, in patients with acquired resistance to third generation TKI is an important requirement for best practice NSCLC treatment.
Materials and Methods: ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide (RS-oligo) complementary to the wild-type sequence. Due to the inherent sequence characteristics of the region between the codons of interest (790 and 797), a RS-oligo focusing on the C797S was developed and is independent of the RS-oligo for T790M, and thus tiling across EGFR Exon 20 can be performed. The RS-oligo in this assay was designed to prevent PCR amplification of wild-type sequences while allowing exponential amplification of any mutations including C797S within the RS-oligo binding region. Serial dilutions of spiked gBlocks® DNA containing C797S mutations mixed with K562 DNA and samples extracted from plasma were amplified with ICE COLD-PCR followed by Sanger sequencing analysis.
Result: For mutations c.2389T&gt;A and c.2390G&gt;C, limits of detection as low as 0.05% were achieved. Forty DNA samples extracted from plasma were also tested. These indicated that the assay is suitable for fragmented samples with low quantities of input DNA such as liquid biopsies.
Conclusion: The pre-amplification PCR product covers both codons 790 and 797. This allows ICE COLD-PCR to be used for the analysis of both T790M and C797S using the same liquid biopsy sample aliquot. This new ICE COLD-PCR method to detect both C797S mutations c.2389T&gt;A and c.2390G&gt;C may provide a highly sensitive, simple and cost effective assay for monitoring acquired resistance to AZD9291 in the liquid biopsies obtained from patients and clinical trials. Coupled with our existing highly sensitive assay for T790M, ICE COLD-PCR offers a highly sensitive method to determine the emergence of resistance mutations, thus potentially contributing to therapeutic optimization.
Citation Format: Grant Wu, Sheena Jensen, Karissa Scott, Erin Montagne, Jason Stoddard, Phil Krzycki, Courtney Schweikart, Benjamin L. Legendre, Philip Eastlake, Katherine A. Richardson. High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1390.