P14.05.B Plasma-EV based liquid biopsy for precision medicine in the treatment of glioblastoma

Abstract

Abstract Background Glioblastoma (GBM) is a devastating and protean brain tumor. Diagnosis and molecular characterization rely on magnetic resonance imaging and brain biopsy which are challenged by low specificity/sensitivity and surgical risks. This hampers the longitudinal monitoring of patients with GBM, whose molecular dynamism ideally requires to refine the therapeutic plan in different disease stages. Extracellular vesicles (EVs) shed by GBM act as a reservoir of circulating biomarkers and can be easily collected through a blood sample, placing the rational to exploit them as a platform for liquid biopsy in GBM. Material and Methods We isolated EVs from 2ml of pre-operative platelet-free plasma by Size Exclusion Chromatography (SEC). EVs are characterized by (i) immunoblot and Fluorescent-Activated Cell Sorter (FACS) for the presence of EV markers; (ii) TEM for morphology and size; (iii) Nanoparticle Tracking Analysis (NTA) for size and concentration; (iv) RNAseq and ddPCR for EV-associated nucleic acid profiling. Results SEC allowed to define EV-enriched fractions expressing the EV-markers CD9, CD81, Flotillin1, lacking the non-EV marker Calnexin and depleted of the majority of plasma contaminants. EV quantification by NTA confirmed their enrichment in the plasma from GBM patients. EV-RNA length (< 200nt) and total yield (1-10ng among different samples) allowed a successful RNAseq library preparation for downstream identification of splicing isoforms, translocations, fused mRNAs, and expression level of GBM-specific transcripts. The ddPCR-based analysis of mutations and copy number alterations proved the ability of cell-derived EV-DNA to recapitulate the alterations of the parental cells. Although the yield of plasma-EV-DNA is low (around 1ng), this paves the way to optimize the isolation/analysis of plasma EV-DNA to permit tumor profiling. Conclusion Plasma-EV based liquid biopsy could implement the personalisation of GBM care for every timepoint of the disease course. Increased plasma-EV concentration is an effective biomarker for GBM presence and its measurement well suits with the clinical routine. EV-RNA/DNA mirror the genomic profile of parental tumor and their analysis could allow the identification of actionable molecular aberrations and the monitoring of GBM mutability during follow-up. Thus, the multilayered analysis of plasma-EV concentration and cargo could enable GBM early diagnosis and to monitor therapy response and the whole tumor evolution.