P05.01.a a Modified Method to Improve the Accuracy of Detecting Glioma Related Gene Abnormalities by Liquid Biopsy

BACKGROUND Current technology has been developed to analyze tumor-derived circulating cell free DNA (ccfDNA) in cerebrospinal fluid (CSF) as a substitute for genomic DNA obtained directly from glioma tissue. However, the amount of ccfDNA obtained from low-grade gliomas and tumors not in contact with the cerebrospinal space is extremely small, making genetic analysis difficult. Here, we report a new assay to identify IDH1 mutations from CSF-derived ccfDNA (below the measurement sensitivity) obtained from patients with low-grade gliomas with IDH1 mutations. MATERIAL AND RESULTS The subjects were five patients with astrocytoma, IDH1-mutant, grade 2 with a confirmed histological diagnosis. CSF was collected by lumbar puncture, extracted with a ccfDNA extraction kit, and DNA was quantified using the Qubit assay. In all cases, the concentration was below the detection limit (0.2 ng/μl). IDH1 mutations could not be detected by the conventional HRM (High Resolution Melting) method. Next, 10 additional cycles of PCR amplification were added to the HRM method, a positive curve indicating a mutation was clearly drawn in all cases, and the IDH1 R132H mutation was confirmed using DNA sequence analysis of the PCR products. By a similar method, it was also possible to analyze H3.3 K27M mutation and BRAFV600E mutation. CONCLUSION Molecular target inhibitors have been developed for IDH-mutated and BRAFV600E-mutated gliomas, and chemotherapy with these inhibitors has become promising. If an assay could be established that can detect gene mutations from cerebrospinal fluid in a minimally invasive manner, even with trace amounts of DNA, it would provide useful information for selecting new treatment methods.