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Updated: Sep 19, 2026
A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications
Published on: September 16, 2022
Implementing multimodal liquid biopsy in glioblastoma: A prospective feasibility study of a standardized clinical
Mario Chiapponi1, Fabiola Cappella1, Laura Andreacchio1
1Department of Neurosurgery, Università Politecnica delle Marche, Azienda Ospedaliero Universitaria delle Marche, Ancona, Italy.
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.
