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Background A Phase 1, single center study is ongoing to evaluate the conditionally replicative oncolytic adenovirus, DNX-2401 (tasadenoturev), followed by radiotherapy (RT) in pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Methods Patients 1–18 years with newly diagnosed DIPG with no prior treatment, Lansky/Karnofsky performance score ≥ 70, and adequate organ function were enrolled. A tumor biopsy was performed followed by a single intratumoral injection of 1e10-5e10 virus particles (vp) DNX-2401. Conventional radiotherapy was initiated within 1 month of DNX-2401 administration. Results Enrolled subjects (n=12) had a median age of 9 (range 3–18) and performance scores of 90–100 (n=4; 33%) or 70–80 (n=8; 67%). As part of a dose escalation design, subjects were treated with 1e10 vp (n=4) or 5e10 vp DNX-2401 (n=8), which was then followed by standard RT in 11 of 12 subjects (92%). No dose-limiting toxicities were observed and the treatment regimen was well-tolerated. Adverse events (AEs) have been primarily mild to moderate and consistent with underlying disease. The most commonly reported AEs (≥ 5 subjects), regardless of study drug relationship, include headache, asthenia, vomiting, anemia, leukocytosis, and fever. Two SAEs have been reported including grade 3 lymphopenia and grade 3 abdominal pain. Tumor reductions have been observed and efficacy evaluations are ongoing. As of 09Dec2020, 12-month survival (OS-12) was 71% and 4 of 12 patients had survived > 20 months. Four subjects continue to be followed for survival. Correlative analysis of tumor biopsy and peripheral samples is ongoing. Conclusions DNX-2401 followed by RT can be safely administered to pediatric subjects with newly diagnosed DIPG; clinical activity and preliminary survival are encouraging.

BACKGROUND Despite our increased understanding of the genetic make-up and new therapies for pediatric high grade glioma (pHGG) and Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains grim. Delta-24-RGD (DNX-2401 in the clinic) has been tested for adult glioblastoma presenting a safe profile and promising efficacy. Recently our group has showed that the virus is safe and effective in preclinical models of pHGG and DIPG. Moreover, we showed that the virus is able to trigger an antitumor immune response. These outstanding preclinical results allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. MATERIALS AND METHODS A phase I clinical trial with DNX-2401 for patients with newly diagnosed DIPG to assess the MTD is taking place in our hospital (N=12). Tumor biopsy is performed through the cerebellar peduncle, followed by virus injection. The virus is injected using a cannula, MEMS cannula (Alcyone Lifesciences) that prevents the reflux. Virus will be injected starting with 1010 pv. The trial is uncontrolled, unicentric with a 3 + 3 design. The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in subjects with DIPG and to collect tumor samples of this type of tumor. Secondary endpoints are overall survival at 12 months (OS12), percentage of responses and induced immune response against tumor. The follow up includes close monitoring of neurological status, blood tests and brain MRI. If this trial shows evidence of safety and efficacy will propel a multicenter clinical trial. RESULTS All the clinical data from the trial available until September 2019 will be presented during the congress, to date 8 patients have been treated within the trial. Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. Patients were home 3–4 days after the injection. All the patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 6 out of the 8 patients. We are currently assessing the immune responses to the virus. CONCLUSIONS Information acquired within this clinical study would aid to understand the response of DIPGs to viral therapies and therefore to better tailor this strategy to improve the survival and the quality of life of pediatric brain tumors.

The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in newly diagnosed DIPG patients (NCT03178032) followed by radiotherapy. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with 1x1010vp and given the lack of toxicity we escalated to 5x1010vp. The procedure was well tolerated and reduced tumor volume was demonstrated in all patients after combined treatment (virus + radiotherapy). We performed molecular studies (RNAseq and the Oncomine Childhood Research Panel from Thermo Fisher). The immune cell composition of the biopsies pre-virus injection was assessed using multiplexed quantitative immunofluorescence. T cells were hardly detectable in these tumors while macrophages were abundant. Using a multiplexed TCR-sequencing mRNA-based assay to analyze 18 available paired pre- and post-treatment samples from the trial, we detected increased clonal T cell diversity following treatment with the virus. We also measured pre and post treatment neutralizing antibodies and their relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). In summary, the virus has shown safety and efficacy in some patients. The information obtained in this clinical study would aid understanding the response of DIPG patients to viral therapies and, therefore, to better tailor this strategy to improve the survival of these patients.

The identification of circulating biomarkers by non-invasive methods would be helpful for glioma detection and response assessment. Strong evidences have shown that GBM cells release microvesicles containing proteins and RNA. We have previously demonstrated that exosomes isolated from the serum of GBM patients had an increased expression of RNU6-1 compared to control samples; therefore it could serve as a non-invasive diagnostic biomarker for GBM. In this study, we set to investigate the role of RNU6-1 as a differential biomarker of GBM versus other brain diseases with similar radiological features. RNU6-1 expression was analysed by digital droplet PCR (ddPCR) in circulating exosomes from serum samples of GBM patients (n=18), healthy controls (n=28), and patients with different brain lesions: subacute stroke (n=30), acute-subacute haemorrhage (n=29), acute demyelinating lesions (n=19), brain metastases (n=21) and Primary CNS Lymphomas (PCNSL) (n=12). We observed that the expression of RNU6-1 was significantly higher in GBM patients (412 ± 550.48 copies/20µL) than in healthy controls (150 ± 224.35 copies/20µL; p=0.039) validating our preceding results. Furthermore, RNU6-1 levels were increased in exosomes from GBM patients than in exosomes from patients with non-neoplastic lesions (stroke [223 ± 709.8 copies/20µL; p=0,067], haemorrhage [127 ± 198.7 copies/20µL; p=0.010], demyelinating lesions [111.5 ± 250.35 copies/20µL; p=0.019]) and PCNSL [18.15 ± 245.7 copies/20µL; p=0.004]). Contrary, RNU6-1 levels were similar between brain metastases and GBM patients [325 ± 632 copies/20µL; p=0.573]. In addition, assessing RNU6-1 as a predictive marker of GBM by ROC curves analysis, we demonstrated that RNU6-1 was a robust diagnostic biomarker of GBM compared to subacute stroke [AUC=0.659; p=0.004], acute/subacute haemorrhage [AUC=0.724; p=0.006], acute demyelinating lesions [AUC=0.728; p=0.011] and PCNSL [AUC=0.814; p

Background: Glioblastoma, the most aggressive primary brain tumor, is genetically heterogeneous. Alternative splicing (AS) plays a key role in numerous pathologies, including cancer. The objectives of our study were to determine whether aberrant AS could play a role in the malignant phenotype of glioma and to understand the mechanism underlying its aberrant regulation. Methods: We obtained surgical samples from patients with glioblastoma who underwent 5-aminolevulinic fluorescence-guided surgery. Biopsies were taken from the tumor center as well as from adjacent normal-appearing tissue. We used a global splicing array to identify candidate genes aberrantly spliced in these glioblastoma samples. Mechanistic and functional studies were performed to elucidate the role of our top candidate splice variant, BAF45d, in glioblastoma. Results: BAF45d is part of the switch/sucrose nonfermentable complex and plays a key role in the development of the CNS. The BAF45d/6A isoform is present in 85% of over 200 glioma samples that have been analyzed and contributes to the malignant glioma phenotype through the maintenance of an undifferentiated cellular state. We demonstrate that BAF45d splicing is mediated by polypyrimidine tract-binding protein 1 (PTBP1) and that BAF45d regulates PTBP1, uncovering a reciprocal interplay between RNA splicing regulation and transcription. Conclusions: Our data indicate that AS is a mechanism that contributes to the malignant phenotype of glioblastoma. Understanding the consequences of this biological process will uncover new therapeutic targets for this devastating disease.

We performed, for the first time, an alternative splicing global analyses (AS) in Glioblastoma (GBM) using specific splicing arrays with paired samples (tumor/non tumor). We identified 51 genes with a significantly different AS and we chose BAF45d as one of the best candidates. BAF45d AS occurs by the exclusion of exon 7 (exon 6a). This exclusion results in a Zinc finger protein domain with the ability to bind specific DNA sequences. The predominant isoform in GBM is one that presents the exclusion of exon 6a (BAF45d-T) and therefore it harbours the zinc finger domain suggesting a possible role in transcription regulation. The inhibition of BAF45d-T in vitro results in a decrease in cell proliferation, in the capacity to self-renew and in the downregulation of markers related with an undifferentiated phenotype. In this line of thinking, we demonstrated that BAF45d-T isoform is present in murine neural progenitor and its expression is reduced during the differentiation toward mature neurons. The inhibition of BAF45d in vivo increases significantly animal survival in a GBM orthotopic model. Moreover, our work revealed that BAF45d splicing was mediated by PTBP1. Interestingly, we also found that BAF45d regulated positively the expression of PTBP1, uncovering a link between splicing regulation and chromatin remodelling. In summary, our results suggest that the AS of BAF45d AS participates in the GBM pathogenesis through the maintenance of an undifferentiated cellular state. In addition, our data suggest that alternative splicing is a mechanism that could be central to GBM development.

BACKGROUND: Immunotherapy is emerging as a promising therapeutic modality for the treatment of glioblastoma (GBM). Recent clinical trials have supported the use of different therapeutic vaccines for high-grade gliomas, demonstrating promising feasibility and safety data. However, the wide variety of approaches and the limitations characterizing most studies preclude firm conclusions regarding efficacy. A phase II trial evaluating the efficacy and safety of autologous dendritic cells vaccination in addition to fluorescence-guided surgery and standard radio-chemotherapy with temozolomide in patients with newly diagnosed GBM was undertaken. PATIENTS AND METHODS: All consecutive patients aged 18 to 70 years who were candidates for resection of a suspected GBM were screened. An attempt at maximum resection was made in every case using fluorescence-guided surgery with 5-aminolevulinic acid. Less than 1 cc of residual tumour and histological confirmation of GBM were required for definite inclusion in the trial. Vaccines were prepared with autologous dendritic cells pulsed with tumour lysate and matured ex vivo. Vaccination calendar started before radiotherapy. Patients also received standard treatment consistent on radiotherapy with concomitant and up to 12 cycles of adjuvant temozolomide. The primary endpoint was progression-free survival (PFS). Secondary endpoints were overall survival (OS), safety, quality of life, and immune response. RESULTS: Thirty-two patients were screened between February 2009 and December 2011. One patient was excluded because of insufficient tumour resection (>1 cc of residual tumour). The mean age of the 31 included patients was 59 years. Median Karnofsky Performance Score (KPS) was 80% (range 60-100%). According to recursive partitioning analysis (RPA), 10% of the patients were RPA class III, 42% were class IV and 48% were class V. O6-methylguanine-DNA methyltransferase (MGMT) promoter was found to be unmethylated in 53% of the patients. Enough tumor lysate and dendritic cells were available in all cases to produce at least 6 vaccine doses. The median number of available vaccine doses was 11,2 (range, 6 to 17). Median PFS was 8.0 months and median OS was 27.4 months (95% CI: 20.9-33.0 months). No severe adverse events attributable to immunotherapy were identified. CONCLUSION: These results suggest that autologous dendritic cells vaccination is feasible and safe, and that the addition of this immunotherapy modality to gross total resection and standard radio-chemotherapy appears to increase survival in patients with newly diagnosed GBM.