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Introduction Precise diagnoses and robust detection of actionable alterations is required for individualized treatments. By using extended molecular diagnostics, the Pediatric Targeted Therapy (PTT) 2.0 program aims at the improvement of diagnostic accuracy and detection of actionable alterations for pediatric high-risk patients. The impact of these analyses on clinical management is reported. Methods Pediatric patients with relapsed or progressive tumors after standard of care treatment were included, independent of histological diagnosis. Formalin fixed paraffin embedded material and a blood sample for germline correction were requested. DNA methylation array, targeted gene panel sequencing (130 genes), RNA and Sanger sequencing in selected cases, and immunohistochemistry (IHC) of selected markers (pERK, pAKT, pS6, PD-L1) were performed. A questionnaire-based follow-up was used to determine the clinical impact of the analysis. Results We enrolled n=263 patients from February 2017 to February 2019. Complete molecular analysis was possible for n=260 cases (99%). The most common entities were brain tumors (n=172/260, 65%). In brain tumors, DNA methylation array alone allowed robust diagnostic classification (score of >=0.9) in n=104/172 cases (60%). Actionable targets as detected by copy number calculation, gene panel sequencing, RNA sequencing and IHC were found in n=94/172 (55%) brain tumor cases. The most common actionable targets in brain tumors were MAPK (pERK, BRAF fusions, BRAF V600E), mTOR (pS6), PI3K (pAKT), CDK4/6 (CDKN2A/B loss), and immune checkpoints (PD-L1). Pathogenic germline alterations with clinical relevance were identified in n=12/172 brain tumor cases (6.9%) and were confirmed by Sanger sequencing, 5/12 (41%) of which were previously unknown. Clinical follow-up of subsequent treatment and outcome are ongoing. Conclusion The combination of next-generation diagnostics such as methylation arrays and targeted sequencing in addition to selected IHC markers added robust information with regard to diagnosis and actionable alterations. The impact on clinical decision-making and on outcome is currently being evaluated.

Background We previously discovered high expression of the disialoganglioside GD2 on H3K27M+ gliomas and demonstrated preclinical efficacy of intravenous (IV) GD2-targeted chimeric antigen receptor (CAR) T-cells in preclinical models of H3K27M-mutated diffuse intrinsic pontine glioma (DIPG) and diffuse midline gliomas (DMGs). We are now conducting a Phase I clinical trial (NCT04196413) of autologous GD2-targeting CAR T-cells for H3K27M+ DIPG and spinal cord DMG. Here we present the results of subjects treated at dose level 1 (DL1; 1 million GD2-CAR T-cells/kg IV). Methods Four patients (3 DIPG, 1 spinal DMG; ages 4–25; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 GD2-CAR T-cells/kg IV on study. One patient with spinal DMG enrolled but became ineligible after manufacturing and was treated on an eIND at DL1. An Ommaya reservoir was placed in all subjects for therapeutic monitoring of intracranial pressure. Subjects underwent lymphodepletion with fludarabine/cyclophosphamide and remained inpatient for at least two weeks post-infusion. Results All subjects developed cytokine release syndrome (Grade 1–3) manifested by fever, tachycardia and hypotension. Other toxicities included ICANS (Grade 1–2) and neurological symptoms/signs mediated by intratumoral inflammation which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred. CAR T cells trafficked to the CNS and were detected in CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and radiographic improvement. The patient treated on an eIND exhibited >90% reduction in spinal DMG volume but progressed by month 3. Re-treatment of this subject via intracerebroventricular administration resulted in a second reduction in spinal DMG volume by ~80%. Conclusions GD2-CAR T-cells at DL1 demonstrate a tolerable safety profile in patients with H3K27M+ DIPG/DMG with clear signs of T-cell expansion and activity including clinical responses.

Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma ({"type":"clinical-trial","attrs":{"text":"NCT03416530","term_id":"NCT03416530"}}NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2, 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0–8 CSF samples and 0–10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Preliminary analysis of samples demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (>1 year) to ONC201, and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to

Leptomeningeal metastatic disease (LMD) occurs in 30–50% of newly diagnosed and recurrent pediatric malignant cerebellar tumors and 20–45% of malignant supratentorial tumors. Radiation and chemotherapy often cause substantial long-term neurotoxicity and outcomes remain poor for patients with LMD. At recurrence, LMD is generally minimally responsive to conventional therapies. Immunovirotherapy with engineered oncolytic HSV-1 G207 has emerged as a promising treatment for children with high-grade brain tumors. G207 infects and kills tumor cells while sparing normal cells and stimulates a robust anti-tumor immune response. Intratumoral G207 inoculation demonstrated safety and preliminary efficacy in a pediatric Phase 1 trial in recurrent/progressive high-grade glioma (NCT02457845), and a Phase 2 trial (NCT04482933) is forthcoming. Additionally, a Phase 1 trial of intratumoral G207 in recurrent/progressive malignant pediatric cerebellar tumors is ongoing (NCT03911388). While intratumoral inoculation delivers G207 directly to a primary tumor, it requires neurosurgical procedures thereby limiting repeat doses. Thus, we sought to establish the safety and efficacy of intraventricular G207. Utilizing an immunocompetent, HSV-sensitive murine strain, we determined that a standard 1x107 plaque-forming units (PFU) dose of G207 resulted in damage to the ependymal lining. However, interferon induction with an intraventricular low-dose (1x104 PFU) of G207 or polyinosinic-polycytidylic acid (poly I:C), a toll-like receptor 3 agonist, three days prior to standard treatment dose protected the ependymal lining. This approach enabled safe delivery of multiple subsequent doses. Importantly, with these protective measures, G207 significantly prolonged survival in pediatric patient-derived xenograft models and an immunocompetent murine LMD model of group 3 medulloblastoma, the most aggressive and fatal subtype. Collectively, these data indicate that toxicity from intraventricular G207 can be safely mitigated prior to a therapeutic dose, and that intraventricular G207 effectively targets group 3 medulloblastoma including LMD. These findings provide support for clinical translation of intraventricular G207.

Background Oncogenic driver alterations in FGFR are present in a subset of pediatric gliomas. Debio1347 is an orally available, highly selective FGFR 1–3 inhibitor that had a favorable safety profile and encouraging preliminary clinical activity in an adult phase 1 study. Methods Five children with progressive/refractory CNS tumors harboring an FGFR gene alteration following prior therapy were treated with Debio1347 at Memorial Sloan Kettering Cancer Center on single patient use protocols. Patients were treated using the 20 mg tablet formulation at the adult recommended phase 2 dose (80 mg/1.73 m2 * BSA once daily). Toxicities were graded using CTCAEv5.0 and imaging response assessments were performed every 8–12 weeks. RESULTS All AEs were grade 1–2. Most common treatment-related adverse events were hyperphosphatemia, ALT increased and hypoalbuminemia (4 patients). Two patients met criteria for partial response and two patients had stable disease. A 13 month-old patient with a spinal cord high-grade glioma harboring two FGFR1 mutations (V592M, K687E) had tumor reduction of 91.7% maintained for 12 months. A 26-month-old patient with a pilomyxoid astrocytoma harboring an FGFR1-TACC1 fusion had a tumor reduction of 74.5% maintained for 9 months. Molecular characterization of recurrent tumor from this patient demonstrated an NF1 deletion as a novel molecular mechanism of acquired resistance to FGFR inhibition. Prolonged disease stabilization was noted in an eight year-old patient with metastatic suprasellar pilomyxoid astrocytoma harboring an FGFR1 mutation (9 months) and in a 14 year-old patient with posterior fossa glioneuronal tumor harboring an FGFR3-TACC3 fusion (24 months and ongoing). Conclusions Debio1347 demonstrated tolerable toxicity and promising anti-tumor efficacy in pediatric patients with refractory FGFR altered gliomas. Specific attention to growth velocity and clinical symptoms with incorporation of imaging assessment of bone growth is warranted. Candidate biomarkers (FGFR1 V592M and K687E SNVs, FGFR-TACC fusions) may guide patient selection. Further studies in this population are warranted.

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no effective therapies beyond radiation. The highly invasive nature of DIPG is key to its aggressive phenotype, but the factors and mechanisms contributing to this aggressive invasion are unknown. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human solid tumors. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, the impact and targetability of ID1 have not been explored in human DIPG. Exome and transcriptome sequencing analyses of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG samples. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChIP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hindbrain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown of primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion/migration and significantly improved survival of K27M-DIPG mice. Knockdown of ID1 in DIPG cells also resulted in down-regulation of the WNK1-NKCC1 pathway, which regulates tumor cell electrolyte homeostasis and migration. Finally, treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 levels, viability of DIPG cells and significantly improved survival of K27M-DIPG mice. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness; and that targeting ID1 with CBD could potentially be an effective therapy for DIPG.

Tumor mutational burden (TMB) has been studied across numerous cancer types as a means of risk stratification. To examine the prognostic relevance of TMB to pediatric central nervous system (CNS) tumors, we conducted a retrospective analysis of patients at Albany Medical Center diagnosed from 2012 to present. Patients were

Recently, sequencing of diffuse intrinsic pontine glioma (DIPG) biopsy specimens has revealed genomic heterogeneity of these tumors, fueling an interest in individualized, targeted treatment options. The Pacific Pediatric Neuro-Oncology Consortium recently completed enrollment onto a feasibility study PNOC003: Molecular Profiling for Individualized Treatment Plan for DIPG (NCT02274987), in which a multidisciplinary tumor board recommended molecularly-targeted agents based on genomic and molecular profiling of each patient’s tumor. Separately, our group developed the Central Nervous System Targeted Agent Prediction (CNS-TAP) tool, which combines pre-clinical, clinical, and CNS penetration data with patient-specific genomic information to allow for numeric scoring of targeted anticancer agents to objectively evaluate these therapies for use in patients with CNS tumors. We hypothesized that highly-scored agents within CNS-TAP would overlap with the agents recommended by the tumor board in this study. For each PNOC003 participant, we utilized the genomic report to identify actionable alterations and input patient-specific data into CNS-TAP to identify the highest scoring agents. We compared high-scoring agents within CNS-TAP with recommendations from the PNOC003 tumor board for each of the enrolled 28 subjects. Overall, 93% (26/28) of patients had at least one agent recommended by both the tumor board and CNS-TAP. Additionally, 38% (37/95) of all agents recommended by the tumor board were also selected by CNS-TAP. Furthermore, we identified factors that likely contributed to the discordance between these two methods. Without clinician input, CNS-TAP is unable to account for drug-drug interactions, includes only designated anticancer agents, and cannot easily be updated in real time. However, CNS-TAP provides an objective evaluation of targeted therapies, whereas tumor boards are inherently subjective. Given the discordance identified between these methods and the strengths of each, a prospective study incorporating both CNS-TAP and a molecular tumor board for targeted therapy selection in DIPG patients is warranted.

Introduction Rapid access to neurosurgical decisions and definitive management are vital for the outcome of neurocritical patients. There are increased challenges of providing services and to maintain critical infrastructure for rural citizens. The relationship between rurality, marginalization and health outcomes has been identified as associated with higher mortality rates and higher rates of many diseases[G1]. Methods Employing linked administrative databases, we retrospectively analyzed a population based cohort of patients diagnosed with a pediatric brain tumour between 1996 to 2017 in Ontario, Canada. The Ontario Marginalization Index was employed as a surrogate for rurality providing an overall Rurality Index for Ontario (RIO) in addition to the 2016 Ontario Marginalization Index (ON-MARG). Results Of 1457 patients included, 54.0% were male, 277 of whom were diagnosed in infancy (i.e., < 3 years of age). Income quintile was evenly distributed with 11.5% classified as living in a rural area of Ontario. The median[G2] distance to the nearest pediatric neurosurgical hospital was 59.6km. The rurality index score (RIO) was 0 in 38.8% of children with the majority of patients with a RIO score of Conclusion Rurality and social determinants of health of the region pediatric neuro-oncological patients reside were not associated with patient outcome but a trend towards lower follow-up compliance was identified when children were from regions with RIO>39. Implementation of telehealth follow-up for these patients may overcome barrier to clinical follow-up.[G5]

Pediatric glioma response to novel targeted therapy can be heterogeneous on conventional proton (1H) MRI. Sodium concentration, as measured with 23Na MRI in adult brain tumors can provide complementary assessment of tumor proliferation to conventional MRI. However, 23Na MRI pediatric brain tumor studies are lacking. Determine the technical feasibility of performing sodium23Na MRI on pediatric glioma patients. Prospective study of an immunotherapy trial for newly diagnosed and recurrent gliomas (high-grade gliomas, low-grade gliomas, brainstem gliomas) in which participants were imaged with 23Na MRI at 3.0 Tesla. The participants (n=26, 14 males) with median age of 11 years (range = 4–23 years of age) were prospectively evaluated with sodium. 23Na MRI is technically feasible in the pediatric population and can distinguish different types of pediatric gliomas at baseline.

Adequate exposure (effective concentration over time) of a therapeutic agent at its site of action is essential for antitumor efficacy. Given constraints of repeat tissue sampling, non-human primate models predictive of pharmacokinetics in pediatric patients have been utilized to assess central nervous system (CNS) exposure. Assessment of cerebrospinal fluid (CSF) drug levels have been used to extrapolate CNS penetration but the relationship of CSF drug levels with tissue distribution is unclear. Utilizing microdialysis, we previously demonstrated geographic variability of drug permeability across the blood:brain barrier (BBB), but this technique is complex and has a high standard deviation. We, therefore, explored a novel technique, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), to compare plasma, CSF, and tissue drug levels in a terminal non-human primate model. Panobinostat, an HDAC inhibitor in clinical trials for DIPG/DMG, was selected for study as it has previously demonstrated poor CNS tissue penetration but suggested modest clinical activity. Methods Panobinostat (p.o., dose 1.6 mg/kg) was administered to non-tumor bearing primates (n=2). One hour following administration (Tmax), blood and CSF were collected, the animal euthanized, brain and spinal cord extracted, and immediately frozen at -80. Panobinostat distribution was mapped on ex vivo sagittal tissue sections using MALDI MSI. To provide specificity and degree of permeability, anatomical structures were segmented for analysis to determine drug concentrations. Blood, CSF and tissue levels of panobinostat were measured via LC-MS/MS. Results Segmentation analysis revealed quantifiable panobinostat, particularly in the lateral ventricles and choroid plexus, and also in the subventricular zone and brainstem, although the overall panobinostat concentration was below the limit of quantitation in these areas. Conclusions Although not reflected in CSF PK, panobinostat is widely distributed in brain tissue. MALDI MSI allows regional assessment of panobinostat penetration and complements CSF pharmacokinetics.

Background Clear identification of tumor subtype is the main predictor of patient outcome and ultimately what is considered an adequate level of surgical risk. At brain tumor resection, imaging modalities and intraoperative histology often give an ambigious diagnosis, complicating intraoperative surgical decision-making. Here, we report a nanopore DNA methylation analysis (NDMA) sequencing approach combined with machine learning for classification of tumor entities that could be used intraoperatively. Methods We analyzed 50 biopsies obtained from biobanked tissue (43, prospective) or sampled at surgery (7, intraoperative) from 20 female and 30 male patients with a median age of 8 years. DNA was extracted using spin columns, quantified on a Qubit fluorometer and assessed for purity using NanoDrop spectrophotometer. DNA was then barcoded with the Rapid Barcoding kit from Oxford Nanopore technologies and loaded onto a MinION flow cell. Sequencing was performed for 3 hours (intraoperative) and 24 hours (prospective). Raw reads were basecalled using the Guppy algorithm, then fed into a snakemake workflow (nanoDx pipeline). This generated a report showing the copy number profile, genome-wide methylation status and subclassification of the tumor according to the Heidelberg reference cohort. Results Twelve different tumor classes were discovered within our cohort spanning from WHO Grade I to Grade IV. The results generated by NDMA were concordant with standard neuropathological diagnosis in 43 out of 50 cases (86%). Of the discordant cases, six were due to the biological complexity of the tumor and one case was misclassified by the pipeline. NDMA enabled correct subclassification of 6/7 intraop cases within a mean of 129 minutes. Conclusion NDMA can accurately subclassify tumor entities intraoperatively and guide surgical procedures when preoperative imaging and frozen section evaluation are unclear.

Prognosis for pediatric patients with advanced relapsed/refractory (R/R) solid (including central nervous system [CNS]) tumors is poor; targeted therapies achieve response rates of only ~15%. Germ cell tumors and high-grade glioma (HGG) are the most common with KIT mutations; sarcoma and HGG are the most common tumors with platelet-derived growth factor receptor alpha (PDGFRA) mutations. Two-year overall survival is 6 months, and in the EU for adults with unresectable/metastatic GIST harboring a PDGFRA D842V mutation. The objectives of this 2-part phase 1/2 multicenter, open-label study, anticipated to enroll 31 patients from Q3 2021, are to assess avapritinib safety, preliminary efficacy, and pharmacokinetics in pediatric patients with KIT/PDGFRA-mutant solid R/R tumors. Eligible patients are aged 2 to

RNA-based therapies offer unique advantages for treating pediatric brain tumors. However, the systemic delivery remains a major problem due to degradation of unmodified RNA in biological fluids, poor brain accumulation, and poor cancer cell uptake or escape from the endosomal lipid bilayer barrier. While nanoparticle encapsulation can prolong circulation time and facilitate cellular uptake, their accumulation in brain tumor remains particularly poor due to their low permeability across the blood-brain barrier and limited intratumoral penetration. Focused ultrasound, when combined with circulating microbubbles (MB-FUS) provides a physical method to transiently modulate the brain tumor microenvironment (TME) and improve nanoparticle delivery. Here, we have examined the delivery of siRNA targeting the Smoothened (SMO) pathway, packaged in 50 nm cationic lipid-polymer hybrid nanoparticles (cLPH:siRNA-SMO), combined with MB-FUS in murine SmoA2 sonic hedgehog (SHH) medulloblastoma. At 30 hours after treatment, we observed the depletion of the SMO protein target, responsible for driving SHH medulloblastoma formation and growth, in mice that had received treatment with MB-FUS and cLPH:siRNA-SMO, but not with cLPH:siRNA-SMO alone. We also confirmed that SMO protein depletion was spatially achieved in the tumor regions with detected cLPH:siRNA-SMO using FISH assay, and that there was 15 fold induction of tumor cell apoptosis compared to tumors in mice that had received cLPH:siRNA-SMO alone. The limited induction of apoptosis was observed with either cLPH:siRNA (non-targeting) or MB-FUS and cLPH:siRNA (non-targeting), suggest that the observed apoptosis induction in the SmoA2 model was the direct result of SMO depletion rather than nonspecific effects of MB-FUS or cLPH:siRNA. Our findings provide a paradigm shift in drug delivery in brain tumors, where physical methods and nanotechnology are tuned together to develop rational strategies for the effective delivery of nucleic acids in brain tumors.

Diffuse intrinsic pontine glioma (DIPG) is the lethal high-grade brain tumor in children with no effective treatment options to date. Despite excessive clinical trials, the prognosis remains poor, with a median overall survival (mOS) of less than 1 year. Genomic studies of DIPG tissue have identified highly recurrent mutations in genes encoding histone H3 resulting in the substitution of lysine to methionine at position 27 (K27M), which is found in approximately 80% of DIPG. Recent drug screening studies identified the histone deacetylase (HDAC) inhibitors panobinostat as a highly effective drug against DIPG in vitro. However, due to the poor Blood-Brain Barrier (BBB) penetration of systemic administration, to enhance the delivery of panobinostat to improve treatment efficacy is needed. Focused ultrasound (FUS) has been shown to be able to safely and non-invasively open BBB to enhance drug delivery. Hence, in this study, we hypothesize that FUS-mediated BBBO (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Herein we established the syngeneic DIPG model by intracranially injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) and used FUS and microbubbles to open BBB and enhance the panobinostat delivery. Magnetic resonance (MR) imaging was utilized to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBB-opening is safe and feasible to mice with DIPG tumors by MR imaging and passive cavitation detection. Moreover, this DIPG cell line is very sensitive to panobinostat in in vitro cytotoxicity assay. The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrated FUS could increase the treatment efficacy of panobinostat to DIPG animals may be due to the increase of targeted delivery of systemic panobinostat to DIPG tumors in brainstem.

Purpose Pediatric central nervous system tumors remain a leading cause of cancer-related death in children and adolescents. Safe sampling of tumor tissue for diagnostic purposes may be challenging. Subclinical detection of disease prior to clinical or imaging progression may provide opportunity for earlier intervention and ultimately improve overall survival. Additionally, our understanding of molecular evolution in response to therapy remains limited, given the rarity of serial sampling of tumor tissue. Methods We report our experience with minimally invasive molecular diagnostics using a validated next generation sequencing assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA) obtained at the time of surgery, by intraventricular catheter or lumbar puncture. All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. Results We analyzed 64 CSF samples from 45 pediatric and adolescent and young adult (AYA) patients (pediatric=25; AYA=20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n=10), medulloblastoma (n=10), pineoblastoma (n=5), low grade glioma (n=4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n=4), metastatic retinoblastoma (n=4), ependymoma (n=3), and other (n=5). Somatic alterations were detected in 28/64 samples (44.4%) and in at least one sample per unique patient in 22/45 patients (48.8%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (86.3%). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient’s disease course. Conclusion We identified four general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: 1) diagnosis; 2) identification of actionable alterations; 3) track response to therapy; and 4) monitoring tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population that may improve care.

Background Cerebellar mutism (CM) is a condition characterized by a significant lack or loss of speech in children following posterior fossa (PF) surgery. The biological origin of CM remains largely unclear and remains the subject of ongoing debate. Despite multidisciplinary rehabilitative interventions, the outcome is less favorable than initially described. Given the treatment refractory nature of CM, central to its management is prevention. Methods A national multi-centered retrospective review of all the children undergoing posterior fossa resection at 4 Canadian academic pediatric institutions was undertaken. Patient, tumour, surgical features suggested to be associated with the post-operative development of CM were reviewed to identify pre-operative and intra-operative factors that may predict post-operative CM occurrence. Results 258 pediatric patients were identified after posterior fossa lesion resection. Mean age at surgery was 6.74 years (SD 4.60) and 42.2% were female. Frozen section was available in 90.3% of cases. The majority of final tumour histology was medulloblastoma (35.7%), pilocytic astrocytoma (32.6%), ependymoma (17.1%) and exophytic glioma (1.2%). Intra-operative impression of adherence to the floor of the 4th ventricle was negative in 47.7%, positive in 36.8% of cases. The extent of resection assessed intraoperatively as gross total resection was 69.8% of cases. Intra-operative abrupt changes in blood pressure and/or heart rate was identified in 19.4% and 17.8% of cases. CM was experienced in 19.5% of patients (N=50), with the majority of cases identified by post-operative day 7. The clinical resolution of CM as mainly assessed by a neurosurgeon (86%) and was complete, significantly resolved, slight improvement, no improvement or deterioration in 56.0%, 8.0%, 20.0%, 14.0%, 2.0% respectively. Conclusion As a devastating surgical complication, identifying and understanding the biological origin of CM is the first step to complication avoidance. Maximal safe resection irrespective of intra-operative pathology remains the goal to avoid the devastating complication of CM.

Patients with medulloblastoma, the most common malignant pediatric brain tumor. need improved treatment options. Conventional medulloblastoma treatment, with resection, chemotherapy, and radiation, leaves survivors at risk of neurocognitive injury, growth defects, and psychosocial impairment. Moreover, there is no effective therapy for recurrent medulloblastoma. We seek to identify novel treatments that will address systemic toxicity and tumor recurrence. We tested a nanoparticle formulation of the CDK4/6 inhibitor, palbociclib (POx-palbo) in mice genetically-engineered to develop medulloblastoma (G-Smo mice) and found a significant anti-tumor effect that was consistently limited by the recurrence of OLIG2+ medulloblastoma stem cells. We then tested the hypothesis that directly targeting OLIG2 function would improve the efficacy of palbociclib and forestall resistance. We therefore examined the potential efficacy of CT-179, a first-in-class OLIG2 inhibitor, in G-Smo mice engineered to express luciferase as an SHH reporter. These studies showed that CT-179 decreased SHH signaling and prolonged event-free survival. Pharmacodynamic studies of G-Smo mice during treatment showed that CT-179 alters cell-cycle progression and promotes a shift towards cell-cycle arrest. Mechanistically, CT-179 decreased the Olig2 phosphorylation. The combination of CT-179 and POx-palbo increased event-free survival of G-Smo mice compared to either agent administered alone. Our studies show both the potential of the palbociclib CT-179 combination, and the potential for CT-179 to improve the efficacy of therapies limited by OLIG2+ stem cell recurrence. As OLIG2+ stem cells have been shown to drive recurrence to conventional therapy and OLIG2 phosphorylation disables p53-driven apoptosis, CT-179 may be a versatile agent to enhance both targeted and cytotoxic treatments.

Introduction Brain tumours are the biggest cancer killer in children and young adults. Several recent developments have the potential to change the treatment of brain tumours in children. These include ultrasound-mediated blood-brain barrier disruption, convection enhanced delivery, polymer delivery systems and electric field therapy, as well as intra-arterial, intra-CSF and intra-nasal chemotherapy. To date, there have been very few clinical trials to evaluate any of these. The science and technology underlying these developments is not traditionally embedded within the standard paediatric neuro-oncology network. In addition, custom-built hardware, novel surgical procedures and, in some cases, the testing and licensing of implantable devices, add difficulty at the regulatory level. Methods The authors participated in an international workshop funded by the charity Children with Cancer UK in 2016, where different experimental techniques aimed at optimising CNS drug delivery were discussed. Following this workshop and two subsequent workshops run by the CBTDDC (Children’s Brain Tumour Drug Delivery Consortium) in 2018 and 2020, the CBTDDC and the recently developed ITCC (Innovative Therapies for Children with Cancer) brain tumour group started working together to set up a new initiative. This aims to develop CNS-delivery-focused trial working groups for paediatric brain tumours. Results We have assembled a prestigious steering group, comprising international researchers and clinicians with expertise in diverse aspects of translational and clinical research in CNS drug delivery. At our first group meeting in March, participants will discuss the most effective ways of translating the emerging drug delivery modalities into clinical trials. Prioritised actions will be taken forward and the group will reconvene to discuss developments and next steps at a workshop in the Autumn. Conclusion We present this abstract to the SNO Paediatric conference to raise awareness of this initiative with the large number of relevant stakeholders who will be attending the event.

Background Patients with relapsed CNS malignancies or DIPG face terrible prognoses. We hypothesized that T cells specific for 3 tumor-associated antigens (TAA), WT1, PRAME and survivin, would be safe and elicit anti-tumor immunity. Methods Patients (n=18) received autologous tumor antigen-associated T cells (TAAT) (up to 8x107/m2) for newly diagnosed DIPG (Group A) or recurrent CNS malignancies (Group B) on a Phase I dose-escalation study (NCT03652545) and were monitored for safety and response. Results/Discussion 16/18 patients who received TAAT completed the 45-day safety monitoring phase with no dose-limiting toxicities. Adverse events were minimal despite multiple pretreatments in Group B. Infused cells were predominantly CD3+ T cells (median 96%; range: 87–99%), with CD4+ and CD8+ comprising 16% (range: 5–87%) and 40% (range: 4–67%) respectively. Specificity for 1–3 TAAs was demonstrated in 11/18 TAAT by a-IFN-γ ELISPOT. Dose escalation is complete, and clinical and immunologic response assessments are ongoing. Plasma cytokine and proteomic analyses demonstrated dynamic post-infusion immune cytokine and protein responses. Consistent with an infusion-mediated immune response all patients in Grp A showed increased T cell effector, inflammatory and immune-stimulatory cytokines IFN-γ, TNF-α, IL-2, IL-5, IL-7, IL-1β, IL-6, IL-8, IL-12p70, IL-17A and GM-CSF at Weeks 1 and 2 post-infusion (n = 6). Of 9 patients who have been tested, 29/92 plasma proteins showed significant differences between dose levels 1 and 2, including increased IL-7 (p

Medulloblastoma, one of the most frequent malignant pediatric brain tumors, encompasses four molecularly and clinically distinct cancers. Sonic hedgehog (SHH)-subtype medulloblastoma constitutes about 30% of medulloblastomas, and therapies targeting the SHH pathway can lead to new highly selective treatment. The haloacid dehalogenase (HAD) phosphatase Eyes Absent 1 (EYA1) is critically involved in the development and progression of SHH-medulloblastoma: Eya1 is highly expressed in SHH-medulloblastomas, and single cell sequencing indicates that Eya1 is a consistent feature that can be detected in every individual cancer cell. Inhibition of EYA1 interrupts SHH pathway signaling. During normal development, EYA1 promotes symmetric division of cerebellar granule cell precursors (GCPs), the cells of origin for SHH-subtype medulloblastoma, and reduced levels of EYA1 decrease medulloblastoma mortality rates in mouse models. Therefore, targeting EYA1 may be a novel therapeutic avenue for these pediatric cancers. Benzarone derivatives have been suggested as allosteric EYA-inhibitors, and benzarone provides a promising platform for chemical derivatives. Here, we develop 60 novel benzarone derivatives and assess their efficacy in inhibiting SHH-medulloblastoma growth through the inhibition of EYA1. Several of the new compounds inhibit EYA1 phosphotyrosine phosphatase activity in a cell-based assay, interrupt SHH pathway, and prevent SHH-medulloblastoma growth in vitro. Our results show that these novel benzarone derivatives are a new promising avenue for developing therapeutics for pediatric SHH-medulloblastoma via inhibition of EYA phosphatases.

Introduction The cytomegalovirus (CMV) antigen, pp65, is ubiquitously expressed in malignant glioma and medulloblastoma but not in healthy brain. The objective of this Phase I trial (NCT03299309) was to assess the safety and feasibility of a novel pp65 peptide vaccine (PEP-CMV) in children and young adults with recurrent medulloblastoma and malignant glioma. Methods Vaccines contain a synthetic long peptide (SLP) of 26 amino acids encoding multiple potential class I, class II, and antibody epitopes of CMV pp65 across several haplotypes. This SLP is administered as an emulsion in Montanide ISA 51. Patients receive a single course of temozolomide to induce lymphopenia, tetanus/diphtheria toxoid site preconditioning, then vaccines administered intradermally every two weeks for 3 doses, then monthly. Results To date, 22 patients have been enrolled. Diagnoses include medulloblastoma (n=2), glioblastoma (n=12), anaplastic oligodendroglioma (n=2), anaplastic astrocytoma (n=3), and malignant glioma NOS (n=3). Mean number of prior treatment regimens is 4.9 (range 1–12). Mean age is 22yo (range 6–35) and 45% of patients are male. The median KPS is 80. The median number of vaccines given at time of analysis is 3.3 (range 1–12). There have been no ≥ 3 Grade toxicities related to the vaccine. One patient developed nausea, vomiting, palpitations, and tachycardia after vaccination and had elevated inflammatory cytokines consistent with cytokine release syndrome. Median PFS is 2.5 months (95% CI: 1.7,4.5) and median OS is 6.5 months (95% CI 3.3, 7.9). Immune response to pp65 as determined by ELISpot was found in 75% of patients. On MRI 6 of the 11 evaluable patients have had at least stable disease with three of those having a partial response. Conclusions Preliminary results demonstrate that PEP-CMV is well-tolerated and elicits an immune response in heavily pretreated, multiply recurrent patients. A multi-institutional Phase II trial is scheduled to open fall 2021.

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.

Children with low-grade glioma have excellent survival rates but often suffer from the morbidity of treatment, particularly from cytotoxic chemotherapies. Targeted agents appear to have some activity but the long-term effects of inhibiting normal developmental pathways are unknown. Lenalidomide is an oral immunomodulatory agent with additional properties including anti-angiogenesis. Phase I studies indicated greater tolerability of this agent compared to adults, and a potential dose-response effect. We performed a Phase 2 trial of lenalidomide in children with pilocytic astrocytoma and optic pathway gliomas who failed initial therapy. The primary objective was to determine the objective response rate of children randomized to Regimen A low-dose (20 mg/m2 /dose) or Regimen B high-dose (115 mg/m2 /dose) lenalidomide, each administering lenalidomide daily x 21 days of each 28-day course. Secondary objectives included estimation of event-free survival (EFS) in this population and correlation of plasma lenalidomide concentration with toxicity and outcome. Results 74 eligible patients were enrolled (n=37 to each arm). The pre-defined activity level of interest was achieved for both arms. Objective responses were observed in both arms, with 4 partial responses in each. A total of n=18 patients completed 26 courses of therapy (Arm A, n=12, Arm B, n=6) The median number of courses on each arm was 14 (range 2–26) for Arm A and 11 for Arm B (range 1- 26). Of the 74 eligible patients who received study drug, 30 required a dose reduction for toxicity (Arm A, n=6, Arm B, n=24) and 16 discontinued treatment on protocol due to toxicity (Arm A, n=2, Arm B, n=14). Conclusion Lenalidomide demonstrates a sufficient level of activity in children with low-grade glioma to warrant further exploration in Phase 3 studies. Low-dose (20 mg/m2) lenalidomide appears to have better tolerability.