Detection of IDH1 and IDH2 Mutation in Formalin-fixed Paraffin-embedded Gliomas Using Allele-specific COLD-PCR and Probe Melting Curve Analysis

Abstract

Many tumors, especially gliomas, contain an isocitrate dehydrogenase (IDH) mutation that can be used for clinical diagnosis and prognosis. Our study aimed to develop a new reliable detection assay for IDH1 and IDH2 mutations for clinical diagnosis based on the allele-specific (AS) coamplification with lower denaturing-polymerase chain reaction (COLD-PCR) and probe melting curve analysis (PMCA). The method includes 3 elements allowing for the sensitive detection of low-abundance mutations: (1) PCR amplification of the target fragments with AS primers; (2) COLD-PCR; and (3) PMCA for differentiating the different mutations after amplification. We conducted a blinded study with 45 paraffin-embedded gliomas specimens and 13 fresh specimens screened for IDH mutations using Sanger sequencing. Concordance between the results of our AS-COLD-PCR/PMCA assay and Sanger sequencing was 100%. Our assay appeared to be superior to direct sequencing with a much higher sensitivity of 0.4% mutations. In summary, our assay is a cost-effective, convenient, and sensitive method for detecting IDH mutations and could be applied in the clinical setting to assess small brain biopsies.