COLD-PCR HRM: A HIGHLY SENSITIVE DETECTION METHOD FOR IDH1 MUTATION
Résumé
Isocitrate dehydrogenase 1 R132H (IDH1R132H) mutation is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature-PCR (COLD PCR), combined with high resolution melting (HRM) for the detection of IDH1R132H mutation. To this end, we used serial dilutions of mutant DNA with wild type DNA. PCR-HRM assay detects IDH1R132H mutation at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild type DNA, that mimicks a tumor sample highly contaminated by normal DNA. We then analyzed ten biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1R132H was positive in 3 cases (30%), whereas double COLD PCR HRM was positive in the ten cases studied (100%). In summary, COLD PCR HRM analysis is 100-fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue.
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