BIOM-32. EVS CARRYING TENASCIN-C ENABLE TUMOR-SPECIFIC MUTATION ANALYSIS IN GLIOBLASTOMA PATIENTS

Abstract Extracellular vesicles (EVs) transport biological and specific information from tumors into the bloodstream, enabling non-invasive detection of tumor material and disease monitoring. Based on a proteomics screen, we performed immunophenotyping of eight glioma-related antigens (tenascin-C (TNC), integrin-beta 1 (ITGB1), profilin-1 (PFN1), CD44, CD133, GPNMB, HLA-II, SPARC) and tetraspanins (CD9, CD63 and CD81) in plasma EVs from GBM patients (before and after surgery (n = 38)), from matched GBM relapse patients (n = 11), and from healthy donors (HD, n = 12) using imaging flow cytometry. Double-positive TNC+/CD9+ EVs showed the strongest differences per mL of plasma in primary (FC = 7.6, p< .0001, ROC analysis AUC = 81%) and relapsed GBM (FC = 16.5, p< .0001; AUC = 90%) compared to HD subjects. High TNC signals were also observed in GBM-EVs by immunogold electron microscopy compared to HD-EVs. In paired analysis, TNC+/CD9+ EVs showed a 3.9-fold decrease after tumor removal (p< .001) and re-increased at GBM recurrence in these patients (FC = 8.4, p< .05; AUC = 84%). In tissue samples, TNC levels were 5.4-fold higher in GBM patients than in non-neoplastic cortex controls (p< .01) measured by immunohistochemistry and correlated positively with plasma TNC+/CD9+ EV levels in RTK-I/II GBM patients (r = 0.42, p< .05). Accordingly, spatial transcriptomics of GBM tissue sections revealed that TNC is specifically overexpressed in GBM cells. Furthermore, magnetic sorting of TNC in plasma EVs allowed detection of GBM-specific TERT*C228T mutations by digital droplet PCR. Mutated TERT DNA was enriched in TNC+ EVs (n = 13) compared to TNC- EVs (FC = 110, p< 0.0001), total EV DNA (FC = 36.7, p< 0.01), and cfDNA (FC = 7.2, p < 0.05). In conclusion, we identified TNC as a biomarker in circulating EVs from GBM patients that can be isolated and used for tumor-specific mutation analysis.