Abstract Introduction Glioma genetic profiling is essential for accurate diagnosis, prognosis, and therapeutic stratification. When tumor tissue is inaccessible, liquid biopsy (LB) offers a non-invasive alternative; however, its utility in glioma is limited by the blood-brain barrier, which restricts the release of tumor DNA into bloodstream. In brain tumors, cerebrospinal fluid (CSF) has been proposed as a more suitable alternative to plasma. This study investigates the use of CSF and plasma-derived extracellular vesicles (EVs) and Nanoparticles (NP) in patients with newly diagnosed malignant glioma, a context in which feasibility and reliability of LB remain controversial. Methodology Glioma tissue samples were collected from two patient cohorts. In cohort 1 (n=45), peritumoral CSF and plasma were also collected, while in cohort 2 (n=39) CSF was obtained via lumbar puncture (LP-CSF). Cell-free DNA (cfDNA), and tumor DNA were analyzed using an NGS panel, droplet digital or BEAMing PCR. Correlations between clinical features and CSF DNA characteristics were examined, and molecular diagnosis based on LB was compared with integrated histopathological diagnosis. To explore the use of plasma EVs as a source of tumor DNA, we (i) verified the release of DNA-containing EVs and NPs from GBM stem like cell cultures, (ii) fractionated EVs and NPs from plasma of GBM patients, and (iii) isolated and analyzed DNA by ddPCR. Results Peritumoral CSF exhibited abundant, often high-molecular weight DNA, whereas LP-CSF typically contained only small quantities of DNA, insufficient for NGS but suitable for targeted testing of IDH, TERT promoter, EGFR, CDKN2A/B, and MGMT methylation (“ITEC” protocol). This enabled tissue-agnostic glioma classification according to WHO2021 in 60% of cases. Notably, absence of tumor DNA in LP-CSF correlated with favorable prognosis. Plasma cfDNA analysis was largely uninformative. GBM stem-like cells release DNA in all EV fractions, with a particular enrichment in NPs, which allow detection of tumor mutations in more than 50% of cases. Conclusions: Our study highlights both the potential and limitations of CSF liquid biopsy for glioma molecular diagnosis at clinical presentation using a flexible and cost-effective approach. It proposes a flexible protocol to extract sufficient diagnostic information from CSF DNA and presents preliminary evidence that plasma nanoparticles may be leveraged for minimally invasive glioma monitoring. Citation Format: Carla Boccaccio, Francesca Orzan, Francesca De Bacco, Elisabetta Lazzarini, Giovanni Crisafulli, Angelo Dipasquale, Marco Macagno, Pasquale Persico, Federico Pessina, Beatrice Bono, Laura Giordano, Pietro Zeppa, Luca Bertero, Paola Cassoni, Antonio Melcarne, Diego Garbossa, Armando Santoro, Paolo M. Comoglio, Stefano Indraccolo, Matteo Simonelli. Glioblastoma liquid biopsy: Maximizing insights from DNA traces in cerebrospinal fluid and blood plasma 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 (6Suppl): Abstract nr A055.
Boccaccio et al. (Mon,) studied this question.
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