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Glioblastoma (GBM) is the most common type of glioma and is uniformly fatal. Currently, tumour heterogeneity and mutation acquisition are major impedances for tailoring personalized therapy. We collected blood and tumour tissue samples from 25 GBM patients and 25 blood samples from healthy controls. Cell-free DNA (cfDNA) was extracted from the plasma of GBM patients and from healthy controls. Tumour DNA was extracted from fresh tumour samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We also collected 180 tumour DNA datasets from GBM patients publicly available at the TCGA/PANCANCER project. These data were analysed for mutations and gene-gene fusions that could be potential druggable targets. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL

In murine experimental glioma models, TLR3 or TLR9 activation of microglial/macrophages has been shown to impair glioma growth, which could, however, not been verified in recent clinical trials. We therefore tested whether combined TLR3 and TLR9 activation of microglia/macrophages would have a synergistic effect. Indeed, combined TLR3/TLR9 activation augmented the suppression of glioma growth in organotypic brain slices from male mice in a microglia-dependent fashion, and this synergistic suppression depended on interferon β release and phagocytic tumor clearance. Combined TLR3/TLR9 stimulation also augmented several functional features of microglia, such as the release of proinflammatory factors, motility, and phagocytosis activity. TLR3/TLR9 stimulation combined with CD47 blockade further augmented glioma clearance. Finally, we confirmed that the coactivation of TLR3/TLR9 also augments the impairment of glioma growth in vivo. Our results show that combined activation of TLR3/TLR9 in microglia/macrophages results in a more efficient glioma suppression, which may provide a potential strategy for glioma treatment. SIGNIFICANCE STATEMENT Glioma-associated microglia/macrophages (GAMs) are the predominant immune cells in glioma growth and are recently considered as antitumor targets. TLRs are involved in glioma growth, but the TLR3 or TLR9 ligands were not successful in clinical trials in treating glioma. We therefore combined TLR3 and TLR9 activation of GAMs, resulting in a strong synergistic effect of tumor clearance in vitro, ex vivo, and in vivo. Mechanisms of this GAM-glioma interaction involve IFNβ signaling and increased tumor clearance by GAMs. Interfering with CD47 signaling had an additional impact on tumor clearance. We propose that these signaling pathways could be exploited as anti-glioma targets.

Gliomas account for about 80% of all malignant brain tumours. Diagnosis is achieved by radiographic imaging followed by tumour resection, to determine tumour cell type, grade and molecular characteristics. Glioblastoma multiforme (GBM) is the most common type of glioma, and is uniformly fatal. The median survival of treated GBM patients is 12-15 months. Standard modalities of therapy are unselective and include surgery, radiation therapy and chemotherapy, while precision medicine has yet to demonstrate improvements in disease outcome. We therefore selected GBM as a model to develop a precision medicine methodology for monitoring patients using blood plasma circulating cell-free DNA (cfDNA). Currently, tumour heterogeneity, clonal diversity and mutation acquisition are the major impedances for tailoring personalized therapy in gliomas in general, and particularly in GBM. Thus, a liquid biopsy diagnostics platform based on cfDNA sequencing may improve treatment outcome for GBM patients, by guiding therapy selection. In this study, we processed from 27 patients with glioma, 27 plasma samples for cfDNA isolation and 5 tissue biopsy samples for tumour DNA isolation. From a control group of 14 healthy individuals, 14 plasma samples were processed for cfDNA isolation. In glioma patients, cfDNA concentration was elevated compared to controls. Point mutations found in glioma tissue biopsies were also found in the cfDNA samples (95% identity). Finally, we identified novel chimeric genes (gene-gene fusions) in both tumour and cfDNA samples. These fusions are predicted to alter protein interaction networks, by removing tumour suppressors and adding oncoproteins. Indeed, several of these fusions are potential drug targets, particularly, NTRK or ROS1 fusions, specifically for crizotinib analogues (like entrectinib and larotrectinib) with enhanced penetration of the central nervous system. Taken together, our results demonstrate that novel druggable targets in gliomas can be identified by liquid biopsy using cfDNA in patient plasma. These results open new perspectives and abilities of precision medicine in GBM.

BACKGROUND GBM is characterized by intratumoral heterogeneity. Tumor heterogeneity, clonal diversity and mutation acquisition hamper the ability to tailor personalized therapy for GBM. Tumor sampling has limited ability to accurately capture the molecular landscape of the tumor and to disclose acquired molecular aberrations. Mutation analysis of cfDNA is a non-invasive procedure which may overcome these limitations as it may reflect the real composition of the tumor and track the molecular evolution. We sequenced cfDNA of GBM patients and assessed mutation patterns and fusion genes. METHODS We collected blood and respective tumor samples from 27 GBM patients and blood samples from 14 healthy controls. Tumor DNA, cfDNA and WBC DNA were sequenced using deep sequencing procedures. The data were analyzed for detection of single nucleotide polymorphism (SNPs) and gene-gene fusions. RESULTS GBM cfDNA concentrations were significantly elevated (median: 23.63 ng/mL; range 12.6–137) compared to healthy controls (median 2.06; range 1.68–7.62) (p < 0.0001). We identified unique SNPs in each glioma patient’s cfDNA and the corresponding tumor DNA including the top-10 most frequently mutated genes in GBM. For example, mutation of TP53 was detected in18.75%; EGFR in 37.5%; NF1-12.5%; LRP1B-25% and IRS4 in 25%. For gene-gene fusion we used the in-house fusion gene database, ChiTaRS 5.0, and identified fusions in cfDNA and tumor DNA. Thus, KMT2A-FLNA was the most frequent fusion found in 16.4% of samples. BCR-ABL1 in 8.82% and FGFR1-BCR in 2.94%. Other fusions included COL1A1-PDGFB (5.88%), NIN-PDGFRB (5.88%), KIF5B-RET (5.88%) and also TPM3-ROS1(2.94%), TFG-ALK(2.94%), MSN-ALK (2.94%) and NPM1-ALK (2.94%) which may be targeted by brain penetrating drugs that are ROS1 and ALK inhibitors. CONCLUSIONS Our study suggests that plasma cfDNA analysis may help to uncover real time mutational and gene fusion status of GBM by a non-invasive procedure. It may identify drug targets based on personalized gene-gene fusions.