Implementing Multimodal Liquid Biopsy in Glioblastoma: A Prospective Feasibility Study of a Standardized Clinical Workflow

Abstract Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, characterized by poor prognosis and limited treatment options. Liquid biopsy offers a minimally invasive approach for molecular profiling and disease monitoring, potentially overcoming tissue sampling and neuroimaging limitations. This prospective, single-center study aimed to establish and evaluate the feasibility of a standardized clinical liquid biopsy protocol for GBM, integrating circulating tumor DNA (ctDNA) and microRNA (miRNA) analyses across multiple biological matrices. Methods Between 2022 and 2024, 26 patients were enrolled, including 14 with histologically confirmed IDH-wildtype GBM, 6 with other intracranial tumors, and 6 with hydrocephalus. Peripheral blood, lumbar cerebrospinal fluid (CSF-L), intraoperative sulcal CSF (CSF-S), and tumor tissue were collected when available. ctDNA was analyzed using a targeted amplicon-based sequencing panel, assessing variant allele frequency and fragmentation index. A panel of GBM-associated miRNAs was quantified by RT-qPCR. Tumor, necrotic, and edema volumes were measured by manual segmentation on contrast-enhanced T1-weighted and T2-FLAIR MRI. Results ctDNA analysis was feasible across all matrices but showed higher rates of suboptimal samples compared with miRNA analysis. Plasma and CSF-L provided comparable ctDNA concentrations, with plasma emerging as the most consistently available matrix. miRNA profiling was robust across matrices, with consistent detectability between CSF-L and CSF-S. Conclusions This study demonstrates the feasibility of integrating multimodal liquid biopsy and volumetric imaging into routine neurosurgical practice for GBM. The proposed workflow provides a reproducible foundation for larger studies aimed at advancing clinical implementation of liquid biopsy strategies.