P14.01.a liquid biopsy of glioma-related genes using a novel amplification technique

Abstract BACKGROUND In the pathological classification of brain tumors, newly revised in 2021, molecular genetic analysis of gliomas has become more important for diagnosis and understanding of the pathogenesis of gliomas. Gliomas are prone to recurrence and malignant progression, and collecting tumor tissue during every genetic analysis is highly invasive. Therefore, techniques have been developed to analyze tumor-derived circulating cell-free DNA (ccfDNA) in the cerebrospinal fluid as a substitute for tissue collection. However, the amount of ccfDNA derived from the cerebrospinal fluid is extremely small and the number of cases for whom genetic analysis is possible is currently limited. In this study, we attempted to establish a novel method for the integrated analysis of glioma-related genes by in vitro amplification of cerebrospinal fluid-derived ccfDNA from glioma patients and a highly sensitive assay. Methods : Tumor-derived ccfDNA was extracted and purified from 1 ml of cerebrospinal fluid obtained by lumbar puncture using the Maxwell RSC instrument in five glioma cases. Hot spots of mutations in IDH1 and H3F3A genes were analyzed by high resolution melting (HRM), TERT mutation by droplet digital PCR, MGMT promoter methylation by methylation-specific HRM, and chromosome 1p/19q loss by multiplex ligation-dependent probe amplification. For samples in which the amount of ccfDNA was too small to analyze, DNA was amplified approximately 500-fold by DNA polymerase using the GenomiPhi kit and analyzed by the same assay method. Results and CONCLUSION s : Although two of the five cases required amplification with the GenomiPhi kit, it was possible to analyze all cases for glioma-related genes as described above. Although the amount of ccfDNA obtained from low-grade gliomas and gliomas that are not in contact with the ventricles or subarachnoid space is small, this amplification method may increase the possibility of liquid biopsy using cerebrospinal fluid in gliomas.