MPTH-01ESTABLISHMENT OF A RAPID AND EFFICIENT GENETIC ANALYSIS SYSTEM OF GLIOMAS

Abstract

PURPOSE: Of the genetic mutations seen in gliomas, IDH1/2 mutations, chromosome 1p/19q loss, MGMT promoter methylation, and genetic alterations of TERT promoter, BRAF, and H3F3A, have become increasingly important as biomarkers for the diagnosis, prognostication, and treatment response of gliomas. Here we report on the establishment of a system, within our institution, of the efficient and rapid detection of these genetic mutations. METHODS: Tumor-derived DNA and RNA extracted from either frozen or paraffin-embedded histological sections were used as specimens. For the point mutation assay, the following mutations of: IDH1/2, H3F3A, BRAF codon 600, and the TERT promoter were screened using High Resolution Melting (HRM) method, and identified by DNA sequencing. For the MGMT promoter methylation, a quantitative analysis was performed using the methylation-specific HRM method. Real-time PCR was used for determining the presence or absence of the BRAF-KIAA fusion gene, and FISH was used for detecting chromosome 1p/19q loss. Further, immunostaining was performed to analyze loss of ATRX (alpha-thalassemia/mental-retardation syndrome-X-linked) expression. RESULTS: Screening of each of the genetic mutations by the point mutation assay using the HRM method was completed in one run of approximately 70 minutes. Furthermore, all genetic mutation results were obtained within 5 ∼ 72 hours after nucleic acid extraction using our assay system. CONCLUSION: For clinics that specialize in diagnosis and treatment of brain tumors, the genetic biomarker information mentioned above will become indispensible in the future. Outsourcing this testing has issues relating to cost and time to obtaining results, and thus testing in-house is desirable. Utilizing PCR primer settings that allow the screening of multiple genetic mutations in one run, and simple and sensitive/specific assays such as the HRM method, allows efficient and rapid analysis.