Abstract Background Pediatric high-grade gliomas (pHGGs), including diffuse midline glioma (DMG) and diffuse intrinsic pontine glioma (DIPG), are the leading cause of central nervous system tumor-related morbidity and mortality in children. Neuronal activity promotes pHGG growth; one key mechanism is neuronal activity-regulated shedding of neuroligin-3 into the glioma microenvironment, mediated by protease ADAM (A Disintegrin and Metalloprotease) 10. INCB007839, an ADAM 10 inhibitor, was used in this trial to block neuroligin-3 shedding and thus strategically target the tumor microenvironment that promotes glioma progression. Study objectives were to determine safety, tolerability and recommended phase 2 dose. Methods Patients aged 3-21 years old with recurrent/progressive pHGGs, including DMG and DIPG, were eligible. One dose level (120mg/m2/dose twice daily [BID]) was tested, and the trial was amended to require prophylactic anticoagulation with enoxaparin due to a thrombotic toxicity. Results Twelve of 13 subjects were eligible, and 10 were evaluable for dose-confirming. Median age was 14.5 years (4.9-20.7 years). Diagnoses included: DIPG (n = 7, 58.3%), glioblastoma multiforme (n = 4, 33.3%), and anaplastic astrocytoma (n = 1, 8.3%). All patients were treated at DL1 and remained on study for 1-4 courses. The most common toxicities were lymphopenia, transaminitis, and fatigue. Nine patients progressed/relapsed during active treatment, 1 died on treatment, and 2 withdrew (1 prior to therapy). Conclusions INCB007839 was tolerated in the majority of patients at 120mg/m2/dose BID with concurrent prophylactic anti-coagulation, although MTD was not declared. These data, combined with foundational preclinical studies targeting neuron-cancer interactions, open the door to possible cancer neuroscience strategies for pediatric high-grade gliomas.

Lay Summary Most children with diffuse intrinsic pontine glioma (DIPG) unfortunately die of their disease. Radiation therapy is the most effective treatment to date, but its effects are temporary and the tumor will eventually begin to grow again. Information from the International DIPG Registry (IDIPGR) regarding childen with DIPG who had radiation a second time was evaluated; this included average age, dose of radiation, use of steroids and the reasons a second course of radiation therapy was given. Our data demonstrates that, in this cohort of children with DIPG who received a second course of radiation, their survival was longer than what has been previously seen with radiation being given only once and this is consistent with another study (by Janssens)1. While significant, it is unclear if children who are able to receive a second course of radiation did better after their first course of radiation, aren’t as ill as others and can tolerate a second course, or otherwise were able to receive it while others did not. More work remains to be done to investigate this further.

Abstract BACKGROUND Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG), are a leading cause of central nervous system tumor-related morbidity and mortality in children. Neuronal activity promotes growth of HGGs; one key mechanism is neuronal activity-regulated shedding of neuroligin-3 into the glioma microenvironment, mediated by the protease (A Disintegrin and Metalloprotease) ADAM10. ADAM10 inhibition slows tumor growth in preclinical pHGG models. Here, we report results of the clinical trial PBTC-056 (NCT04295759) evaluating safety and tolerability of INCB007839, an inhibitor of the ADAM 10 and 17 proteases. METHODS Patients aged 3-21 years old with recurrent/progressive pHGGs, including DIPG, were eligible. Additional eligibility criteria included measurable disease and failure of at least 1 standard treatment. One dose level (120mg/m2/dose twice daily [BID]) was tested, and the trial was subsequently amended to require prophylactic anticoagulation with enoxaparin due to an unanticipated toxicity. The primary objective was to assess the safety and tolerability of INCB007839 for children with pHGGs. RESULTS 12 of 13 eligible subjects were evaluable. Median age was 13.5 years (4.9-20.7 years). Diagnoses included: DIPG (54%), glioblastoma multiforme (31%), anaplastic astrocytoma (8%) and CNS primary tumor NOS (8%). All patients were treated at DL1 and remained on study for 1-4 courses. The most common toxicities were lymphopenia, elevated transaminases, and fatigue. There were 3 dose-limiting toxicities: Grade 5 cerebral venous thrombosis (n=1), Grade 3 alanine aminotransferase increase (n=1) and Grade 2 thrombocytopenia (n=1). Ten patients progressed/relapsed during active treatment, 1 patient died on treatment, and 2 patients withdrew (1 prior to starting therapy). CONCLUSIONS INCB007839 was generally well tolerated with a recommended phase 2 dose of 120mg/m2/dose BID and concurrent prophylactic anti-coagulation. These data, combined with foundational preclinical studies targeting neuron-cancer interactions, open the door to possible cancer neuroscience strategies, including combination therapy, for pediatric high-grade gliomas.

Abstract BACKGROUND FLAG-003 is a novel, rationally-designed, CNS-penetrant, dual-acting agent that inhibits receptor tyrosine kinase activity and microtubule polymerization. Preclinical studies in glioblastoma (GBM) demonstrated decreased cell viability, reduced tumor volumes, decreased microvessel density, and increased survival. Remarkably, using an orthotopic DIPG model, all mice treated with FLAG-003 at 275 mg/day/orally displayed tumor regression, tumor reduction and increased survival. Given the significant preclinical activity against DIPG, as well as the known heterogeneity of DIPG, we evaluated the efficacy of FLAG-003 in vitro against a panel of DIPG cell lines. METHODS Eight DIPG cell lines hosted at the Center for Patient Derived Models (CPDM) were selected based on genomic and growth characteristics. Cells were plated on 384-well plates; 24 hours after plating, cells were dosed with 1nM to 50uM (12 step log curve) FLAG-003. Three technical replicates were performed per step. Controls consisted of positive kill control Staurosporine, negative growth control of 0.5% DMSO, or no DMSO for baseline growth. ONC201, a small molecule antagonist of DRD2/3 currently in clinical trials for DIPG, was used as a comparator. We also evaluated two GBM patient-derived models. RESULTS FLAG-003 demonstrated efficacy in all DIPG and GBM cell lines tested with IC50 less than 100nM. FLAG-003 was more potent compared to ONC201 in all models evaluated. CONCLUSIONS Single agent FLAG-003 demonstrated activity against a panel of DIPG cell lines. As next steps, we are exploring the efficacy of this drug in orthotopic DIPG PDX models. Continued development of FLAG-003 as a potential therapeutic candidate for DIPG patients is warranted and underway.

Background: The frequency and significance of IDH mutations in glioma across age groups is incompletely understood. We performed a multi-center retrospective age-stratified comparison of patients with IDH-mutant gliomas to identify age-specific differences in clinico-genomic features, treatments, and outcomes. Methods: Clinical, histologic, and sequencing data from patients with IDH-mutant, grade 2-4 gliomas, were collected from collaborating institutions between 2013-2019. Patients were categorized as pediatric (<19y), YA (19-39y) or older adult (>= 40y). Clinical presentation, treatment, histologic, and molecular features were compared across age categories using Fisher's exact test or analysis-of-variance. Cox proportional-hazards regression was used to determine association of age and other covariates with overall (OS) and progression-free survival (PFS). Results: We identified a cohort of 379 patients (204 YA) with IDH-mutant glioma with clinical data. There were 155 (41%) oligodendrogliomas and 224 (59%) astrocytomas. YA showed significantly shorter PFS and shorter median time-to-malignant transformation (MT) compared to pediatric and adult groups, but no significant OS difference. Adjusting for pathology type, extent of resection, and upfront therapy in multivariable analysis, the YA group was independently prognostic of shorter PFS than pediatric and adult groups. Among astrocytomas, CDK4/6 copy number amplifications were associated with both shorter PFS and shorter OS. Among oligodendrogliomas, PIK3CA and CDKN2A/2B alterations were associated with shorter OS. Conclusions: IDH-mutant glioma YA patients had significantly shorter PFS and time to MT but did not differ in OS compared to pediatric and adult groups. Treatment approach varied significantly by patient age and warrant further study as addressable age-associated outcome drivers.

Abstract BACKGROUND Preclinical studies of diffuse intrinsic pontine glioma (DIPG) have increased significantly over the past 15 years due to the development of DIPG disease-specific tools including cell lines and animal models. We now know that DIPG is a molecularly heterogeneous tumor. While >80% of these tumors have H3K27M alterations, partner mutations can vary. Many DIPG cell lines used for pre-clinical assessments of new therapies have not been fully characterized, therefore, comparing preclinical results across studies has been impeded. We sought to Create, Characterize and Catalogue DIPG cell lines and animal models with the aim of expanding contemporary resources and providing a public database for DIPG researchers. METHODS Existing DIPG cell lines in the Center for Patient-Derived Models (CPDM) were utilized; additional patient-derived cell lines were created in CPDM, and obtained from collaborators. The assembled collection was characterized by whole genome (WGS) and RNA-sequencing (RNAseq). RESULTS To date, we have 21 growth-verified DIPG models with the majority grown as 3D neurospheres in-vitro and capable for long-term culture. Fourteen (14) of these patient-derived models were genomically verified with WGS and RNA-seq platform. Data will be made publicly available to researchers on cBioPortal. CONCLUSIONS Systematic characterization and cataloguing of pre-clinical DIPG models is important to allow more stringent comparison across studies and ensure their utility and impact. These DIPG-specific tools can thus be used in applications such as drug discovery and testing.

The role of permanent cerebrospinal fluid (CSF) diversion in the management of patients with diffuse intrinsic pontine glioma (DIPG) is poorly elucidated. There are no standard practice guidelines regarding the role of these neurosurgical interventions in patients with DIPG or large studies regarding the impact of CSF diversion on overall survival (OS) or palliation in this disease. Data were extracted from subjects registered in the International DIPG registry (IDIPGR). Univariable analyses were performed using the Fisher’s exact test or Wilcoxon rank sum test. Survival was estimated using the Kaplan-Meier method. Of n=1104 evaluable patients with DIPG registered in the IDIPGR, n=303 (27.4%) had permanent CSF diversion. Median time from diagnosis to CSF diversion was 1 month (range 0-4 months). There were no significant differences in age, gender or race in subjects with CSF diversion versus no CSF diversion. Patients with CSF diversion had a higher incidence of hydrocephalus at diagnosis (64.1% vs 11.3%, p<0.001). There was no significant difference in OS or post-progression survival (PPS) at 1,2 and 3 years between subjects with permanent CSF diversion compared to those without (p=0.5 and p=0.8, respectively). Median OS was 11 months in both. Median PPS was 7 months (no CSF diversion) and 6 months (CSF diversion). Patients reported less headache and vomiting after permanent CSF diversion compared to pre-diversion (p<0.0001), however steroid use was also significantly higher after CSF diversion (p<0.001). In this, the largest reported international cohort of patients with DIPG with permanent CSF diversion, we show that permanent CSF diversion was not associated with an improvement in OS or PPS. CSF diversion improved headache and vomiting but was also associated with increased rates of steroid use. This highlights the need for consensus guidelines regarding the use of these diversion strategies in patients with DIPG.

Abstract BACKGROUND Given that radiation therapy is the only treatment modality demonstrated to result in any clinical benefit for children with DIPG, re-irradiation therapy has been explored as a treatment option for progressive DIPG. Several studies suggest re-irradiation is feasible, and may lengthen survival for children with progressive DIPG. However, any re-irradiation benefits are unclear and no standard of care (dose, fractionation, volume, timing, clinical status) has been defined. The aims of this study are to evaluate re-radiation therapy practices for children with progressive DIPG/DMG and to define a historical cohort of children with DIPG/DMG who have received re-radiation (re-XRT) for progressive DIPG/DMG. METHODS Data was extracted from the International DIPG Registry, and analyzed with descriptive statistics. RESULTS Of 1214 patients in the iDIPG Registry, 113 receiving re-XRT. Patients were diagnosed between 2002-2022, with the majority of patients diagnosed over the last decade. Of those 113 patients with specified data, at re-irradiation, n=68 (60%) received photon and n= 4 (4%) were treated with proton radiation. The median dose at re-XRT was 25 Gy (20-30 IQR) in 10 fractions (10-14 IQR). Time between initial XRT and re-XRT was 41.5 weeks (29.5-54 weeks). OS from diagnosis was 18 months (range, 15-23 mo); OS from re-XRT was 6 months (range, 4-10 mo). CONCLUSIONS Re-radiation therapy for children with DIPG/DMG is becoming a more common practice and appears to have OS benefit, with post-progression OS of 6 mo comparing favorably to historical data of 2.3 mo. [Cooney T et al, Neuro Oncol 2017] Efforts are underway to define patient selection and tolerability. Data extraction from the SIOPE DIPG/DMG Registry is also ongoing; data from both registries will be collated and uniformly presented.

Vebreltinib (APL-101) is a potent c-Met inhibitor which has shown efficacy in murine pre-clinical models and is under clinical trial evaluation for several cancer types, including adult high-grade glioma. The capsule formulation (APL-101-Cap) was reformulated as a pill (APL-101-Pill) with the expectation of increasing bioavailability. We determined and compared the plasma pharmacokinetics (PK) and cerebrospinal fluid (CSF) penetration of APL-101-Cap and APL-101-Pill in a non-human primate (NHP) model. METHODS: Seven animals, previously developed as serial CSF access models, were utilized across all studies. One NHP was studied with both formulations. Both formulations were administered orally as a single drug/single dose in serial studies, followed by serial plasma and CSF sample collections from 0-96 hours. Dosages: APL-101-Cap 3.25 mg/kg (n = 3) or 6.5 mg/kg (n = 4) and APL-101-Pill 5 mg/kg (n = 4). The resulting plasma concentrations from each formulation were quantified by LC-MS/MS. PK parameters were calculated via noncompartmental methods, and results were analyzed to assess CNS penetration (AUCCSF:AUCPlasma) and compare formulations. RESULTS: Mean (range) for plasma and CSF PK exposure [AUCLAST (hr*ng/mL)] reported. APL-101-Cap plasma exposure, regardless of dose, was more variable than APL-101-Pill. APL-101-Pill had lower exposure in both plasma (AUC 1893.0 (841-2865)] and CSF [AUC 27.7 (5.0-65.0)] than APL-101-Cap regardless of dose [(3.25 mg/kg): plasma AUC 9621.0 (1331-23132) and CSF AUC 688.0 (24-1352); 6.5 mg/kg: plasma AUC 6208.0 (1424-19800) and CSF AUC 606.0 (34-1179)]. CNS penetrance was higher with APL-101-Cap (3.2% [0.5-5.8%] at 3.25mg/kg; 4.2% (2.4-6%) at 6.5 mg/kg), but also more variable when compared to APL-101-Pill (1.2% [0.6-2.3%]). For APL-101-Cap, one animal at 3.25 mg/kg and two animals at 6.25 mg/kg had undetectable CSF concentrations. CONCLUSION: In this NHP model, single-dose oral administration of APL-101-Pill resulted in lower, but less variable plasma and CSF exposure compared to APL-101-Cap. APL-101-Cap CSF exposure was unaffected by dose escalation.

Midline glioma patient demographic information, and samples analyzed for genomic and ctDNA studies.

Assessing specificity of ddPCR platform by testing non-CNS malignant pediatric CSF and plasma.

BackgroundRecent data indicates that cerebrospinal fluid (CSF) dynamics are disturbed after stroke. Our lab has previously shown that intracranial pressure rises dramatically 24 h after experimental stroke and that this reduces blood flow to ischaemic tissue. CSF outflow resistance is increased at this time point. We hypothesised that reduced transit of CSF through brain parenchyma and reduced outflow of CSF via the cribriform plate at 24 h after stroke may contribute to the previously identified post-stroke intracranial pressure elevation.MethodsUsing a photothrombotic permanent occlusion model of stroke in C57BL/6 adult male mice, we examined the movement of an intracisternally infused 0.5% Texas Red dextran throughout the brain and measured tracer efflux into the nasal mucosa via the cribriform plate at 24 h or two weeks after stroke. Brain tissue and nasal mucosa were collected ex vivo and imaged using fluorescent microscopy to determine the change in CSF tracer intensity in these tissues.ResultsAt 24 h after stroke, we found that CSF tracer load was significantly reduced in brain tissue from stroke animals in both the ipsilateral and contralateral hemispheres when compared to sham. CSF tracer load was also reduced in the lateral region of the ipsilateral hemisphere when compared to the contralateral hemisphere in stroke brains. In addition, we identified an 81% reduction in CSF tracer load in the nasal mucosa in stroke animals compared to sham. These alterations to the movement of CSF-borne tracer were not present at two weeks after stroke.ConclusionsOur data indicates that influx of CSF into the brain tissue and efflux via the cribriform plate are reduced 24 h after stroke. This may contribute to reported increases in intracranial pressure at 24 h after stroke and thus worsen stroke outcomes.

BACKGROUND:Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood cancer with median survival of less than 1 year. Panobinostat is an oral multihistone deacetylase inhibitor with preclinical activity in DIPG models. Study objectives were to determine safety, tolerability, maximum tolerated dose (MTD), toxicity profile, and pharmacokinetics of panobinostat in children with DIPG.PATIENTS AND METHODS:In stratum 1, panobinostat was administered 3 days per week for 3 weeks on, 1 week off to children with progressive DIPG, with dose escalation following a two-stage continual reassessment method. After this MTD was determined, the study was amended to evaluate the MTD in children with nonprogressive DIPG/Diffuse midline glioma (DMG) (stratum 2) on an alternate schedule, 3 days a week every other week in an effort to escalate the dose.RESULTS:For stratum 1, 19 subjects enrolled with 17/19 evaluable for dose-finding. The MTD was 10 mg/m2/dose. Dose-limiting toxicities included thrombocytopenia and neutropenia. Posterior reversible encephalopathy syndrome was reported in 1 patient. For stratum 2, 34 eligible subjects enrolled with 29/34 evaluable for dose finding. The MTD on this schedule was 22 mg/m2/dose. DLTs included thrombocytopenia, neutropenia, neutropenia with grade 4 thrombocytopenia, prolonged intolerable nausea, and increased ALT.CONCLUSIONS:The MTD of panobinostat is 10 mg/m2/dose administered 3 times per week for 3 weeks on/1 week off in children with progressive DIPG/DMG and 22 mg/m2/dose administered 3 times per week for 1 week on/1 week off when administered in a similar population preprogression. The most common toxicity for both schedules was myelosuppression.

Supplementary figures 1-4 and supplementary table 3. Supplementary figure 1. Genomic DNA validation of probes, assessing sensitivity, and specificity of ddPCR platform. Supplementary figure 2. Novel detection of histone 3 mutation in fluid present in a brainstem tumor cyst found in a DIPG patient at postmortem. Supplementary figure 3. Longitudinal changes in plasma ctDNA in association with MR imaging findings and clinical assessments. Supplementary figure 4. Biofluid ctDNA and tumor spread as assessed by MRI, genomic, and/or histological studies. Supplementary table 3. Mutations analyzed in the study by ddPCR with corresponding design of primers and probes.

Clinical information for PNOC003 plasma ctDNA studies.

PURPOSE Children with low-grade glioma often require long-term therapy and suffer from treatment morbidity. Although targeted agents are promising, tumor targets often encompass normal developmental pathways and long-term effects of inhibition are unknown. Lenalidomide is an immunomodulatory agent with wide-ranging properties. Phase I studies indicated greater tolerability of lenalidomide in children compared with adults and a potential dose-response effect. PATIENTS AND METHODS We performed a phase II trial of lenalidomide in children with pilocytic astrocytomas and optic pathway gliomas who failed initial therapy. Primary objectives included determination of objective response rate of children randomly assigned to regimen A, low-dose (20 mg/m 2 /dose), or regimen B, high-dose (115 mg/m 2 /dose) lenalidomide, and assessment for early progression. Secondary objectives included estimation of event-free survival, overall survival, incidence of toxic events, and assessment of plasma lenalidomide concentrations. Lenalidomide was administered once daily × 21 days of each 28-day cycle for each regimen. RESULTS Seventy-four eligible patients were enrolled (n = 37, each arm). The predefined activity level of interest was achieved for both arms. Four objective responses were observed in each arm, and the number of early progressors was low. Eighteen patients completed 26 cycles of therapy (regimen A, n = 12; regimen B, n = 6). The median number of cycles was 14 (range, 2-26) for regimen A and 11 for regimen B (range, 1-26). Of 74 eligible patients who received study drug, 30 required dose reduction for toxicity (regimen A, n = 6; regimen B, n = 24) and 16 discontinued because of toxicity (regimen A, n = 2; regimen B, n = 14). CONCLUSION Lenalidomide demonstrates a sufficient level of activity in children with low-grade glioma to warrant further exploration. Low-dose (20 mg/m 2 /dose administered once daily × 21 days of each 28-day cycle) lenalidomide appears to have better tolerability with comparable activity.