Detection of the IDH1/2 Gene Mutations in Tumor Samples with Low-Abundance Mutant Allele

Objective: Identification of driver mutations in tumors is an extremely important task in oncology for the choice of treatment strategy and assessment of therapy efficacy. In many cases, especially in disease monitoring, there is a need to detect a small number of copies of the mutant allele against the background of excessive content of wild-type DNA. Methods: Genomic DNA was isolated from tumor tissue in paraffine blocks and amplified using polymerase-chain reaction (PCR) with blocking of wild-type DNA amplification via addition of locked-nucleic acid (LNA) oligonucleotides. Fluorescently labelled PCR-product enriched by IDH-mutant alleles was hybridized on a biochip with immobilized oligonucleotide probes which was able to determine 5 mutations in the IDH1 gene and 2 mutations in the IDH2 gene. Results and Discussion: The method was developed and tested on a collection of 26 samples of paraffinized tumor tissue (glioma, glioblastoma, chondrosarcoma). In three cases, R132C, R132L, and R132H mutations in the IDH1 gene were detected in tumor samples with low representation of the mutant allele. The limit of detection of mutant DNA was determined to be 0.1% in the wild-type DNA background. The advantages of the method are simultaneous analysis of multiple targets, simplicity, reliability, and cost-effectiveness. Conclusions: A highly sensitive method for detecting somatic mutations in the IDH1/2 genes by LNA-mediated blocking of amplification of wild-type alleles and hybridization in a biological microchip was developed. We believe that the method may be useful for detecting low-abundance mutations in tumor samples, as well as in circular tumor DNA.