Abstract A048: A modified method to improve the accuracy of detecting glioma-related gene abnormalities for pediatric gliomas by liquid biopsy

Abstract Background Analysis of tumor-derived circulating cell-free DNA (ccfDNA) in cerebrospinal fluid (CSF) has emerged as a minimally invasive alternative to tissue-based genomic profiling for gliomas in both pediatric and adult populations. However, the amount of detectable ccfDNA is often extremely limited in low-grade gliomas and in tumors without direct contact with the cerebrospinal fluid space, posing a major challenge for molecular diagnosis. This issue is particularly pronounced in pediatric and adolescent and young adult (AYA) patients, in whom repeat tumor biopsy is often difficult. Here, we report a modified assay to detect glioma-associated gene mutations (IDH1 and BRAFV600E) from CSF-derived ccfDNA below the conventional measurement sensitivity. Methods and Results 10 patients with histologically or radiologicaly confirmed low-grade astrocytoma were included in this study. CSF samples were obtained by lumbar puncture, and ccfDNA was extracted using Maxwell (Promega) kit. DNA concentration was quantified using the Qubit assay. In all cases, the DNA concentration was below the detection limit (0.2 ng/μl), and IDH1 or BRAF mutations could not be detected using the conventional High Resolution Melting (HRM) method. By incorporating an additional 10 cycles of PCR amplification prior to HRM analysis, a clear positive melting curve suggestive of mutation was observed in all cases. Next, gene mutations were subsequently confirmed by DNA sequence analysis of the PCR products. In samples obtained tumor tissue, the analysis results of tissue-derived and ccfDNA were consistent. Using the same modified approach, H3.3 mutation were also successfully detected. Conclusion Molecularly targeted therapies have been developed for IDH-mutated and BRAFV600E-mutated gliomas across age groups, and accurate molecular characterization is increasingly important for treatment selection. The modified assay described here enables sensitive detection of clinically relevant gene mutations from cerebrospinal fluid using trace amounts of DNA. This minimally invasive approach may be broadly applicable to both adult and pediatric glioma patients, with particular clinical utility in pediatric and AYA populations, where tissue acquisition is often limited. Citation Format: Masayoshi Fukuoka, Eita Uchida, Junichi Adachi, Ryo Nishikawa, Tomonari Suzuki. A modified method to improve the accuracy of detecting glioma-related gene abnormalities for pediatric gliomas by liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr A048.