Abstract 1829: The validation of a pyrosequencing KRAS mutation detection assay

Abstract

Mutations in the KRAS gene, especially affecting the codon 12 and 13 region of the Kras protein, have implications in the treatment of certain cancer types. Because of the current potential for the FDA to oversee laboratory developed procedures (LPDs), it is imminently important that the validation of such tests be published to ensure that it is shown that these LPDs are properly reviewed and properly tested within the CLIA guidelines, thus keeping LPDs within the CLIA regulatory domain. We have developed a pyrosequencing assay that can detect eleven mutations in the codon 12 and 13 position of the KRAS gene. Our validation consisted of a sensitivity study in which a purified mutant PCR product was introduced into a wildtype DNA background and diluted down to undetectable levels. This sensitivity test was done on six different KRAS codon 12/13 mutants to determine the detection limit of the assay. Intra- and inter-assay precision and assay accuracy was determined by comparing assay results of over 40 total samples, both mutant and wild type, over five days carried out by two technicians. The results will show that this KRAS pyrosequencing assay falls within the acceptance criteria for sensitivity, accuracy and precision. Citation Format: Matthew L. Poulin, Ann Meyer, E. Andrew Mead, Jessica Xu, Ryan Drennan, Liyin Yan. The validation of a pyrosequencing KRAS mutation detection assay. [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 1829.