Abstract 5251: TaqMan® rare mutation assays for QuantStudio® 3D digital PCR system

Abstract

Abstract Detection and quantification of mutant alleles in tumor tissue allow for research disease monitoring and the research of drug efficacy. Detection of emerging secondary mutations in the same tumor tissue causing resistance to potential treatment will help guide decisions on future treatment plans. A less invasive research method than using tumor tissue is testing for the presence of mutations in circulating free DNA (cfDNA). We created a research tool for mutation detection at a sensitivity level of 1% and below. This allows researchers to find correlation between types of mutations and types of tumors and determination of potential secondary mutations. The tool combines TaqMan® SNP Genotyping Assays with digital PCR. A set of assays was optimized for use in digital PCR with the QuantStudio® 3D Digital PCR System. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan® SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. Our initial set of 38 assays covers mutations commonly found in tumor tissues: BRAF V600E, mutations in EGFR exons 19, 20 and 21, KRAS codons 12 and 13, PIK3CA exons 9 and 20, and the JAK2 V617F mutations. All assays were wet-lab tested at a 10% mutation rate and a 1% mutation rate using mutant plasmid spiked into wild-type genomic DNA. Additionally, selected assays were tested at the 0.1% mutation rate using mutant cell lines spiked into wild-type genomic DNA. Wet-lab results confirm that all assays showed superior performance discriminating mutant and wild-type alleles. Mutant alleles were successfully detected as low as 0.1%. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Marion Laig, Brian Ho, Nivedita S. Majumdar, Le T. Lac, Frances Chan, Ramesh Sathiyaa, Iain Russel, Paco Cifuentes, Ted Straub, Kamini Varma, David Keys. TaqMan® rare mutation assays for QuantStudio® 3D digital PCR system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2015-5251