LAUSR

Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are enzymes that normally catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate during glucose metabolism. Heterozygous mutations in the IDH1 and IDH2 genes have been identified in a specific subset of cancers. Mutations typically affect the enzyme active sites at codon R132 in the IDH1 gene, and R140 or R172 in the IDH2 gene. This confers neomorphic enzyme activity that produces the oncometabolite 2-hydroxyglutarate that is involved in cancer initiation and progression. The detection of IDH1/IDH2 mutations has important diagnostic, prognostic, and therapeutic implications and testing for these variants is being incorporated in clinical settings. In fact, the identification of IDH1 or IDH2 gene mutations is now required for the classification of tumors of the central nervous system under the 2016 WHO guidelines. Therefore, there is a significant need for an IDH1 + IDH2 test that can be performed in a rapid manner to support clinical diagnosis and treatment decisions. A strategy utilizing allele-specific quantitative PCR (qPCR) is well suited for evaluating a small number of hotspot variants in IDH1 and IDH2 from clinical tissue samples in a matter of few hours. Using RNase H2-dependent PCR (rhPCR) and a universal reporter system, we have developed new multiplex qPCR assays that effectively discriminate between closely related sequences that differ by only a single nucleotide base at the same position or adjacent position in a single tube reaction. In addition, the rhPCR mechanism of the assays also greatly reduces the impact of primer-primer interactions, further improving the multiplex capability of the assays. During initial testing, specificity, accuracy, and sensitivity were evaluated. Mutations present in patient samples at frequencies as low as 2% were identified with high reproducibility. The assays have been tested across 49 patient samples that included FFPE tissue, blood, and bone marrow aspirate with results that are concordant to those found using an orthogonal method of next generation sequencing. Our study showed that this assay provides a rapid method for detection of multiple IDH1 or IDH2 mutations in single rhPCR reactions with high accuracy, specificity, and sensitivity. Citation Format: Yun Bao, Aymen Baig, Yu Wang, A. John Iafrate, Caifu Chen, Darrell R. Borger. Rapid detection of IDH1/2 mutations using RNaseH2 dependent, multiplex quantitative PCR Assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2017-740