OPTIMIZED APPROACHES FOR LIQUID BIOPSY IN GLIOBLASTOMA PATIENTS

Background Glioblastoma (GBM) remains the deadliest primary brain tumour, with a 5-year survival < 4 % despite maximal multimodal therapy. Its diffuse infiltration, marked spatial-temporal heterogeneity, and the shielding effect of the blood–brain barrier (BBB) limit the accuracy of tissue biopsy and conventional imaging, hampering truly personalised treatment. To overcome these limitations, novel strategies are needed that can non-invasively capture tumour-specific information Objective This thesis explores three complementary, clinically compatible strategies to enhance the informativity of liquid biopsy: plasma extracellular vesicles (EVs), 5-aminolevulinic-acid (5-ALA)–induced protoporphyrin IX (PpIX) fluorescence, and focused-ultrasound blood–brain-barrier opening (FUS-BBBO). The objective is to determine whether they can aid in collecting accurate, real-time information on tumour burden and biology, thereby laying the groundwork for precision monitoring and therapy. Methods We enrolled 66 GBM patients, 83 healthy controls and 50 patients with other CNS lesions for the EV arm; 20 tumour patients and 4 healthy volunteers for the 5-ALA arm; and 4 GBM patients across two FUS trials. Plasma-EVs - EVs were isolated by size-exclusion chromatography and quantified/sized with tunable resistive-pulse sensing technology at different timepoints during disease course. PpIX fluorescence - after oral 5-ALA, paired urine, plasma, and serum samples were analyzed with a dedicated fiber-optic spectrometer developed as part of this study and validated against a FluoroMax reader FUS-BBBO - In four GBM patients enrolled in two MR-guided FUS trials, plasma was collected immediately before and 30–60 min after sonication; EV metrics and cfDNA were measured. Results Plasma-EVs distinguish GBM from healthy and non-GBM cohorts with high accuracy (near-perfect AUC), although brain metastases can overlap with GBM. EV concentrations drop sharply 72 h post-resection and rise at recurrence, sometimes preceding MRI. Mean EV diameter yields an added signature, also relative to brain metastases, that normalizes after surgery. Together, these findings support EV enumeration/size as actionable diagnostic and follow-up biomarkers using SEC/TRPS-compatible hospital workflows. 5-ALA/PpIX read-out -EV-incorporated PpIX was below fluorescence detection thresholds, precluding direct EV identification. Conversely, freely circulating PpIX is quantifiable with a portable fiber-optic probe, with near-perfect linear agreement between platforms. In urine, a spectral index (619/525 nm) robustly discriminates patients from controls, whereas plasma/serum signals were an order of magnitude lower FUS-BBBO enhanced liquid biopsy - Paired pre- and post-procedure plasma samples revealed patient-specific changes in circulating signals (EVs, cfDNA), consistent with preclinical and early-clinical evidence that BBBO modifies tumour-derived analytes under specific conditions. Conclusions Profiling of plasma EVs provides a dual biomarker, based on concentration and size shift, that reflects tumour presence and treatment response, while urinary PpIX fluorescence adds a rapid, non-invasive read-out of tumor presence. FUS-BBBO safely modulates BBB permeability and can elicit tumour-derived analytes changes, highlighting a path to boost blood-borne signals. Collectively, these optimized liquid-biopsy tools may facilitate the implementation of precision oncology in GBM, although confirmation in larger prospective studies is still needed.