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Purpose:PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG).Patients and Methods:Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN).Results:Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome.Conclusions:Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

Supplementary Figure from Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

Background Cross-sectional tumor measures are traditional clinical trial endpoints; however volumetric measures may better assess tumor growth. We determined the correlation and compared the prognostic impact of cross-sectional and volumetric measures of progressive disease (PD) among patients with DIPG. Methods Imaging and clinical data were abstracted from the International DIPG Registry. Tumor volume and cross-sectional product (CP) were measured with mint Lesion™ software using manual contouring. Correlation between CP and volume (segmented and mathematical [ellipsoid] model) thresholds of PD were assessed by linear regression. Landmark analyses determined differences in survival (via log-rank) between patients classified as PD versus non-PD by CP and volumetric measurements at 1, 3, 5, 7, and 9 months postradiotherapy (RT). Hazard ratios (HR) for survival after these time points were calculated by Cox regression. Results A total of 312 MRIs (46 patients) were analyzed. Comparing change from the previous smallest measure, CP increase of 25% (PD) correlated with a segmented volume increase of 30% (R2 = 0.710), rather than 40% (spherical model extrapolation). CP-determined PD predicted survival at 1 month post-RT (HR = 2.77), but not other time points. Segmented volumetric-determined PD (40% threshold) predicted survival at all imaging timepoints (HRs = 2.57, 2.62, 3.35, 2.71, 16.29), and 30% volumetric PD threshold predicted survival at 1, 3, 5, and 9 month timepoints (HRs = 2.57, 2.62, 4.65, 5.54). Compared to ellipsoid volume, segmented volume demonstrated superior survival associations. Conclusions Segmented volumetric assessments of PD correlated better with survival than CP or ellipsoid volume at most time points. Semiautomated tumor volume likely represents a more accurate, prognostically-relevant measure of disease burden in DIPG.

Background Diffuse intrinsic pontine glioma (DIPG) remains a clinico-radiologic diagnosis without routine tissue acquisition. Reliable imaging distinction between DIPG and other pontine tumors with potentially more favorable prognoses and treatment considerations is essential. Methods Cases submitted to the International DIPG registry (IDIPGR) with histopathologic and/or radiologic data were analyzed. Central imaging review was performed on diagnostic brain MRIs (if available) by two neuro-radiologists. Imaging features suggestive of alternative diagnoses included nonpontine origin, Results Among 286 patients with pathology from biopsy and/or autopsy, 23 (8%) had histologic diagnoses inconsistent with DIPG, most commonly nondiffuse low-grade gliomas and embryonal tumors. Among 569 patients with centrally-reviewed diagnostic MRIs, 40 (7%) were classified as non-DIPG, alternative diagnosis suspected. The combined analysis included 151 patients with both histopathology and centrally-reviewed MRI. Of 77 patients with imaging classified as characteristic of DIPG, 76 (99%) had histopathologic diagnoses consistent with DIPG (infiltrating grade II-IV gliomas). Of 57 patients classified as likely DIPG with some unusual imaging features, 55 (96%) had histopathologic diagnoses consistent with DIPG. Of 17 patients with imaging features suggestive of an alternative diagnosis, eight (47%) had histopathologic diagnoses inconsistent with DIPG (remaining patients were excluded due to nonpontine tumor origin). Association between central neuro-imaging review impression and histopathology was significant (p < 0.001), and central neuro-imaging impression was prognostic of overall survival. Conclusions The accuracy and important role of central neuro-imaging review in confirming the diagnosis of DIPG is demonstrated.

OBJECTIVE Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by germline mutations in the TP53 gene. CNS tumors are the fourth most common tumor type in LFS, and recent screening guidelines demonstrate that early tumor detection is associated with improved long-term survival. However, there is a paucity of data regarding surgical intervention when lesions are identified in asymptomatic patients on surveillance imaging. The authors investigated this through their cohort and literature review. METHODS The cohort consisted of children seen in the Pediatric Cancer Genetics Program at Children’s National Hospital between August 2012 and August 2021. The authors also include a PubMed (MEDLINE) literature search of articles from 2006 to 2021 related to surveillance and CNS tumors in patients with LFS. Studies in which CNS tumors were not identified or detailed patient information was not provided were excluded. Patients from the selected articles and the authors’ cohort were added for further analysis. RESULTS Between August 2012 and August 2021, 10 children with LFS and CNS tumors were assessed at Children’s National Hospital: 4 who were known carriers of the TP53 mutation had CNS lesions found on surveillance imaging, whereas 6 presented with symptomatic CNS lesions and were either known or subsequently found to have germline TP53 mutations. The literature search identified 148 articles, 7 of which were included in this review. Patients from the literature and the present cohort were added for a total of 56 CNS lesions. A majority of the low-grade CNS lesions (22/24, 92%) were found on surveillance protocols in asymptomatic patients, whereas the majority of the high-grade lesions (22/26, 85%) presented in symptomatic patients who were not undergoing routine surveillance or as the initial diagnosis of LFS. The authors noted a significant survival advantage in pediatric patients with low-grade lesions, with an overall survival of 100% at 30 months. Minor limitations of the study include patient sample size and limitations in the patient cohort due to this being a retrospective rather than a prospective study. CONCLUSIONS Data presented in this study support surveillance protocols in LFS and demonstrate the importance of dedicated CNS imaging and early surgical intervention when lesions are identified. Systematic review registration no.: CRD42022372610 (www.crd.york.ac.uk/prospero)

Background Recurrent atypical teratoid/rhabdoid tumor (AT/RT) is, most often, a fatal pediatric malignancy with limited curative options. Methods We conducted a phase II study of Aurora kinase A inhibitor alisertib in patients aged Results SD (n = 8) and partial response (PR) (n = 1) were observed among 30 evaluable patients. Progression-free survival (PFS) was 30.0% ± 7.9% at 6 months and 13.3% ± 5.6% at 1 year. One-year overall survival (OS) was 36.7% ± 8.4%. Two patients continued treatment for >12 months. PFS did not differ by AT/RT molecular groups. Neutropenia was the most common adverse effect (n = 23/30, 77%). The 22 patients who received liquid formulation had a higher mean maximum concentration (Cmax) of 10.1 ± 3.0 µM and faster time to Cmax (Tmax = 1.2 ± 0.7 h) than those who received tablets (Cmax = 5.7 ± 2.4 µM, Tmax = 3.4 ± 1.4 h). Conclusions Although the study did not meet predetermined efficacy end point, single-agent alisertib was well tolerated by children with recurrent AT/RT, and SD or PR was observed in approximately a third of the patients.

Purpose: PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Patients and Methods: Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). Results: Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. Conclusions: Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.

BackgroundCraniopharyngiomas account for approximately 1.2–4% of all CNS tumors. They are typically treated with a combination of surgical resection and focal radiotherapy. Unfortunately, treatment can lead to permanent deleterious effects on behavior, learning, and endocrine function.MethodsThe Pediatric Brain Tumor Consortium performed a multicenter phase 2 study in children and young adults with unresectable or recurrent craniopharyngioma (PBTC-039). Between December 2013 and November 2017, nineteen patients (median age at enrollment, 13.1 y; range, 2–25 y) were enrolled in one of 2 strata: patients previously treated with surgery alone (stratum 1) or who received radiation (stratum 2).ResultsEighteen eligible patients (8 male, 10 female) were treated with weekly subcutaneous pegylated interferon alpha-2b for up to 18 courses (108 wk). Therapy was well tolerated with no grade 4 or 5 toxicities. 2 of the 7 eligible patients (28.6%) in stratum 1 had a partial response, but only one response was sustained for more than 3 months. None of the 11 stratum 2 patients had an objective radiographic response, although median progression-free survival was 19.5 months.ConclusionsPegylated interferon alpha-2b treatment, in lieu of or following radiotherapy, was well tolerated in children and young adults with recurrent craniopharyngiomas. Although objective responses were limited, progression-free survival results are encouraging, warranting further studies.

Gliomas are the most common primary central nervous system tumors occurring in children and adults with neurofibromatosis type 1 (NF1). Over the past decade, discoveries of the molecular basis of low-grade gliomas (LGGs) have led to new approaches for diagnosis and treatments. However, these new understandings have not been fully applied to the management of NF1-associated gliomas. A consensus panel consisting of experts in NF1 and gliomas was convened to review the current molecular knowledge of NF1-associated low-grade “transformed” and high-grade gliomas; insights gained from mouse models of NF1-LGGs; challenges in diagnosing and treating older patients with NF1-associated gliomas; and advances in molecularly targeted treatment and potential immunologic treatment of these tumors. Next steps are recommended to advance the management and outcomes for NF1-associated gliomas.

INTRODUCTION Cross-sectional tumor measures are used as endpoints in clinical trials of DIPG, but may not capture meaningful changes in disease burden. Volumetric measures may provide a more accurate assessment of tumor growth. We measured the correlation between cross-sectional and volumetric measures and compared their prognostic impact to better understand response evaluation in DIPG. METHODS Patients from the International DIPG Registry with diagnostic and post-radiation MRIs were included. Utilizing mint LesionTM software, tumors were manually contoured by an experienced pediatric neuro-radiologist to extrapolate cross-sectional product (CP) and volume measures. Correlation between CP and volume was assessed by linear regression. Landmark analyses were performed to determine differences in overall survival (OS) (via log-rank) between patients classified as progressive disease (PD) versus non-PD according to CP and volumetric measurements at one-, three-, and five-months post-radiation. Imaging consistent with pseudoprogression was designated non-PD. Hazard ratios (HR) for survival after these timepoints were calculated by Cox regression. RESULTS A total of 317 MRIs from 46 patients were analyzed. When comparing change from smallest previous tumor size, CP increase of 25% (PD by RAPNO) correlated with volume increase of 28% (R2=0.685). There was no difference in OS between patients classified as PD versus non-PD by CP at one-month, three-months, or five-months post-radiation (p >0.05). However, significant differences in OS were observed between patients classified as PD versus non-PD by volume (28% increase) at one-month (2.7 vs. 12.8 months, p=0.005), three-months (1.9 vs. 10.7 months, p=0.036), and five-months post-radiation (3.7 vs. 9.1 months, p=0.023). PD by volume, but not by CP, was predictive of survival at all timepoints (HR: 5.0, 2.4, 2.4). CONCLUSIONS Volumetric assessments of PD correlated better with survival than CP at all post-radiation timepoints. Tumor volume likely represents a more accurate, prognostically-relevant measure of disease burden that deserves investigation in future DIPG trials.

BACKGROUND Pharmacokinetic modeling for previously reported phase 1A data of pan-RAF inhibitor DAY101 in RAS/RAF/MEK/ERK pathway-altered tumors suggested a correlation between higher doses and improved efficacy without clear safety data. The protocol was amended to explore differential dosing across different body surface areas (BSA). METHODS Eligible patients were < 25 years old with radiographically recurrent/progressive RAS/RAF/MEK/ERK pathway-altered tumors. We applied a novel modification of a TITE-BOIN design to determine recommended phase 2 dosing of oral, weekly DAY101 in evaluable patients within two BSA subgroups: ≤ 1.5 m2 or > 1.5 m2. Target toxicity probability was closest to 20%. We tested 420 and 530 mg/m2. Dose limiting toxicities (DLTs) were determined within Cycle 1. Evaluable patients had either ‘complete’ or ‘partial’ information. Complete information meant patient had a DLT or received 3 of 4 planned doses with no DLT. Partial information meant patient received only 1 or 2 doses or did not complete the DLT observation period. Three patients with complete information at a given dose level were required before dose escalation. The primary endpoint driving dose escalation was time from start of Cycle 1 to first DLT or, if no DLT, time from start of Cycle 1 to minimum of date of last contact or end of Cycle 1. RESULTS We treated 35 eligible patients: 21 KIAA1549:BRAF-, 9 BRAFV600E-, 4 novel RAF- and one FGFR1-altered tumors. Histologically, cohort included 30 LGGs, 4 high grade gliomas and 1 soft tissue sarcoma. There were 6 DLTs: 3 in each BSA subgroup, all at 530 mg/m2/dose, all grade 3, and 5 known side effects ( 2 fatigue, 3 rash, 1 menorrhagia). CONCLUSIONS Oral weekly DAY101 is well tolerated. The TITE model recommends 530 mg/m2/dose PO weekly for patients with BSA < 1.5m2 and 420 mg/m2/dose PO weekly for patients with BSA >1.5m2.

BACKGROUND RAF alterations are oncogenic drivers found in most pediatric low-grade gliomas (LGGs). Tovorafenib is an investigational, oral, selective, CNS-penetrant, small molecule, type II pan‑RAF inhibitor. METHODS FIREFLY-1 (NCT04775485) is a multicenter phase 2 study evaluating the safety and efficacy of tovorafenib monotherapy. Registrational arm 1 enrolled patients with recurrent/progressive LGG harboring an activating BRAF alteration. Patients aged 6 months–25 years who progressed following ≥ 1 prior line of systemic therapy were eligible. Tovorafenib 420 mg/m2 (≤ 600 mg) was administered weekly (tablet or liquid suspension formulation) until progression or for ≥ 26, 28-day cycles. The primary endpoint (arm 1) was overall response rate, as defined by RANO criteria, per independent review. RESULTS As of April 14, 2022, 25 patients were enrolled to arm 1 and had ≥ 6 months of follow-up. Median age at enrollment was 8 years (range 3–18). Most patients had astrocytomas (92%), 48% with optic pathway involvement. Patients were heavily pretreated (56% with ≥ 3 prior lines of therapy), and 72% previously received MAPK pathway-targeted agents. Tumors harbored BRAF fusions (84%) or BRAF V600E mutations (16%). Per independent assessment, partial responses (1 unconfirmed) were seen in 14 (64%) of 22 evaluable patients, with 6 additional patients having stable disease, and a clinical benefit rate of 91%. Responses were achieved in tumors with BRAF fusions and V600E mutations. Most treatment-emergent adverse events (AEs) were grade 1 or 2 (96%). The most common grade ≥ 3 AEs were anemia (12%), vomiting, increased blood creatinine phosphokinase and maculopapular rash (8% each). Seven patients (28%) required dose modification for treatment-related AEs; no patients discontinued tovorafenib due to AEs. Updated results, including efficacy per RAPNO assessments will be presented. CONCLUSIONS Tovorafenib was generally well tolerated and showed encouraging evidence of antitumor activity in children with pretreated BRAF-altered LGG.

BACKGROUND Monitoring for dissemination in medulloblastoma involves spinal MRIs (sMRIs) and lumbar punctures (LPs). The frequency and duration of these studies vary depending on risk of dissemination; general practice varies between and within institutions. Our goal is to review the current practice in post-treatment monitoring via examination of published data, clinical trial standards, and expert practice. METHODS We reviewed the available literature and accessible clinical trial protocols. Using PRISMA guidelines, we conducted searches of Ovid MEDLINE, Embase, and PubMed to identify studies which detailed relapse rates and patterns, and recommended practice for patients with medulloblastoma aged 3-21 years. Eligible studies from 1990 to present were included. RESULTS Thirteen multi-institutional clinical trials from North America and Europe were analyzed for frequency of post-therapy surveillance with LPs/sMRIs. Several studies (n=8) recommend LPs/sMRIs every 3-6 months in standard risk and every 3 months in high risk disease for two years when initially completing therapy, and then often diverge in frequency after 2 years. Some studies (n=5) did not include post-treatment recommendations. Cumulative data on relapse from available clinical trial reports and single institution studies (n=673 relapses) demonstrated local failure rate of 18.4%, distant failure of 49.1%, and combined distant and local failure of 32.5%. Spine-only relapses were reported to occur in approximately 13-20%, with one small study reporting spine-only relapse rate of 37.5%. DISCUSSION Patterns of recurrence in medulloblastoma have shifted over time as therapeutic approaches have evolved, including more spinal-only recurrences. Standardized guidance regarding surveillance could have significant impact on clinical detection and streamline management to minimize unnecessary examinations. Early detection of recurrent disease could optimize treatment options for providers and families, whether focusing on curative approaches or quality of life.

BACKGROUND: The ReMIND trial hypothesizes that autologous T-cells specific for three tumor-associated antigens (TAA) -WT1, PRAME, and survivin- will be safe and elicit anti-tumor immunity in pediatric patients with CNS cancer. METHODS: Patients (n=25) received autologous TAA-T for newly-diagnosed DIPG (Stratum A, up to 4x107/m2) or recurrent CNS malignancies (Stratum B, up to 8x107/m2) in this dose-escalation study (NCT03652545) and were monitored for toxicity and response. RESULTS: Autologous TAAT products were successfully manufactured from 28 patients. Using IFN-γ ELISPOT assay, 10/11 evaluable products had specificity for 1-3 TAAs. 25 patients received TAA-T (6 in Stratum A and 19 in Stratum B) and completed the 45-day safety monitoring period. Twenty-four (96%) had no dose limiting toxicities (DLT), but 1 (4%) patient with DIPG experienced a DLT related to potential immune-mediated pseudoprogression. Median overall survival for patients with DIPG (Stratum A) was 14 months (range, 6-32 months). Median progression-free survival (PFS) for Stratum B patients was 8 months (range, 2-26+ months), which exceeded their preceding median duration of disease stability of 2 months (range, 1-5 months). Plasma cytokine profiles demonstrated infusion-related immune cytokine responses. CONCLUSIONS: In summary, TAAT had a favorable toxicity profile (4%) especially compared to CAR-T therapy and may elicit anti-tumor immune responses that contribute to prolonged survival. Immunobiology studies and response assessments are ongoing for both strata. Based on these encouraging preliminary results, we have added a stratum that includes prescribed lymphodepletion pre TAA-T administration at a cell dose of 8x107/m2. Further, we plan to add an additional stratum to allow direct administration of TAA-T into the CNS via an Ommaya reservoir.

BACKGROUND: There are no standard practice guidelines for cerebrospinal (CSF) diversion for diffuse intrinsic pontine glioma (DIPG), nor clear understanding of potential for palliation and life-prolongation. We evaluated CSF diversion characteristics in children with DIPG to determine incidence, indications, symptom effects, and survival. METHODS: Data were extracted from subjects registered in the International DIPG registry (IDIPGR). Univariable analyses was performed using the Fisher’s exact test or Wilcoxon rank sum test. Survival was estimated using the Kaplan-Meier method. RESULTS: Evaluable patients (n=542) met criteria for DIPG diagnosis by central radiologic review; of those, 126 (23%) had permanent CSF diversion. Median time from diagnosis to diversion was 0.5 months (IQR 0.1-4.5 months). Those with permanent diversion were significantly younger (median 5.4 years vs 7.0 years, p

BACKGROUND RAS/RAF/MEK/ERK pathway activation is the primary driver for most pediatric low-grade gliomas (pLGG). MEK162 (binimetinib) is an orally bioavailable MEK1/2 inhibitor with superior brain penetration in a preclinical model. The primary objective of this multi-institutional phase II and target validation study was to assess stratum-specific efficacy of binimetinib in progressive pLGG. METHODS Eligible children aged 1-18 years with previously treated radiographically progressive pLGG were enrolled and treated with binimetinib, starting dose 32mg/m2/dose twice daily. Stratum 1 included patients with pLGG with documented BRAF fusion; stratum 2, neurofibromatosis 1 (NF1)-associated pLGG; stratum 3, sporadic pLGG without documented BRAF fusion; and stratum 4, patients undergoing planned tumor biopsy who began binimetinib preoperatively. Partial and minor responses (PR and MR) were defined as ≥50% and ≥25% decrease in maximal two-dimensional measurements. RESULTS Of 86 patients enrolled, 85 were evaluable for response. Of these, 48 (56%) showed a radiographic response (30 PR and 18 MR) in the first year of treatment. Response rate for stratum 1 (n=28) was 50% (12 PR and 2 MR); 12 (43%) had stable disease (SD) and 2 (7%) progressive disease (PD). Stratum 2 (n=21) response rate was 43% (5 PR, 4 MR), with 12 (57%) SD and no PD. Stratum 3 (n=29) response rate was 69% (10 PR, 10 MR), 4 (14%) SD and 5 (17%) PD. Stratum 4 (n=7) include 3 PR, 2 MR, 2 SD. Nineteen (22%) discontinued treatment for toxicity (most commonly dermatologic), and an additional 42 (49%) required dose reduction. Median dose at the time of PR/MR was 28mg/m2; responses were seen at doses as low 16mg/m2. CONCLUSION Binimetinib is highly effective in the treatment of both NF1-associated and sporadic pLGG, with or without documented BRAF fusion. Modified dosing strategies to improve tolerability may be considered in future trials.

INTRODUCTION: Panobinostat is an oral HDAC inhibitor with pre-clinical activity against DIPG. The phase I study in children with progressive DIPG (stratum 1) defined the maximum-tolerated dose (MTD) as 10 mg/m2 administered 3x/week, 3 weeks on/1 week off. Herein, we report results of stratum 2, involving children with non-progressive DIPG/DMG using an alternative schedule. Primary objectives were to describe the toxicity profile and define the MTD; secondary objectives were to describe progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: Patients with non-progressive DIPG or H3K27M-mutated thalamic DMG were eligible >14 days following standard radiation therapy only. Panobinostat was given every other day, 3x/week, on alternate weeks. Patients who received at least one dose of panobinostat were evaluable for toxicity. Four dose levels (DL) were evaluated: DL1 (16mg/m2/dose), DL2 (22 mg/m2/dose), DL3 (28 mg/m2/dose) and DL4 (36 mg/m2/dose). Dose escalation was determined by a continuous reassessment method. Correlative studies included pharmacokinetics obtained on course 1, day 1, and day 3 prior to subsequent dosing. RESULTS: Thirty-four eligible patients (median age, 7.6 [3-16] years) were enrolled with 29 evaluable for dose finding; DL1, n=3; DL2, n=10; DL3, n=11; DL4, n=5. The primary toxicities were myelosuppression and gastrointestinal. Eight DLTs occurred: DL2, Grade 3 thrombocytopenia (n=1); DL3, Grade 4 neutropenia (n=3), Grade 4 neutropenia and Grade 4 thrombocytopenia, (n=1); DL4, Grade 2 nausea (n=1), Grade 3 increased ALT (n=1), Grade 4 thrombocytopenia (n=1). Median PFS from drug initiation was 4.4 (1-11.2) months; median OS from diagnosis was 11.7 (4.5-25) months. These did not significantly differ from the PBTC historical cohort (PFS, p-value 0.4967; OS, p-value 0.6457). CONCLUSION: The MTD of panobinostat administered on this schedule to children with non-progressive DIPG/DMG is 22 mg/m2/dose. The primary DLT was myelosuppression. There was no significant improvement in PFS or OS in this cohort.

INTRODUCTION: DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) will implement an innovative clinical trial design of combinatorial arms for patients with DMG at all disease stages, that is adaptive to pre-clinical and correlate data generated in eight collaborating institutions. The goal of the team is to rapidly identify and validate i) promising drugs and drug combinations for clinical use, and ii) predictive biomarkers of promising drugs. METHODS: In vitro (n=30) and in vivo (n=8) models of DMG across fourteen institutions were used to assess single and combination treatment of over 80 drugs and drug combinations. Predictive biomarkers of response for top candidate drugs were identified using extensive molecular assays including proteomics, CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS: Inhibitory concentration (IC50) of all drugs were established and validated across all participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), ONC201+panobinostat (p=0.01). Marizomib was highly toxic in murine PDX and zebrafish larvae assays. Murine pharmacokinetic analysis showed peak brain levels of ONC201, and ONC206 above pre-clinical IC50 concentrations. Molecular testing and analyses of existing drug screen across 578 cancer cells validated mitochondrial stress and additional proteins, as the main targets induces by ONC201/6. CONCLUSION: Thorough preclinical testing in a multi-site laboratory setting identified promising therapeutics for DMGs, resulting in launch of two clinical trials (PNOC022, ONOC023). Validation of identified biomarkers are ongoing using clinical specimen as well as in vivo PDX models.

Background A phase I/II trial of vorinostat (suberoylanilide hydroxamic acid), an oral histone deacetylase inhibitor, was conducted in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) through the Children’s Oncology Group (COG) to: 1) determine the recommended phase II dose (RP2D) of vorinostat given concurrently with radiation therapy; 2) document the toxicities of continuing vorinostat as maintenance therapy after radiation; and 3) to determine the efficacy of this regimen by comparing the risk of progression or death with a historical model from past COG trials. Methods Vorinostat was given once daily, Monday through Friday, during radiation therapy (54 Gy in 30 fractions), and then continued at 230 mg/m2 daily for a maximum of twelve 28-day cycles. Results Twelve patients enrolled in the phase I study; the RP2D of vorinostat given concurrently with radiation was 230 mg/m2/day, Monday through Friday weekly. The six patients enrolled at the RP2D and an additional 64 patients enrolled in the phase II study contributed to the efficacy assessment. Although vorinostat was well-tolerated, did not interrupt radiation therapy, and was permanently discontinued in only 8.6% of patients due to toxicities, risk for EFS-event was not significantly reduced compared with the target risk derived from historical COG data (P = 0.32; 1-sided). The 1-year EFS was 5.85% (95% CI 1.89–13.1%) and 1-year OS was 39.2% (27.8–50.5%). Conclusions Vorinostat given concurrently with radiation followed by vorinostat monotherapy was well tolerated in children with newly diagnosed DIPG but failed to improve outcome.

BACKGROUND H3 K27M-mutant DMG predominantly affects children and young adults; no effective therapy is known. ONC201 is a first-in-class, anti-cancer DRD2 antagonist and ClpP agonist. METHODS Fifty pediatric and adult patients with recurrent H3 K27M DMG who received oral ONC201 monotherapy in clinical trials and expanded access were selected for a planned efficacy analysis. Eligibility criteria included measurable contrast-enhancing disease by RANO-HGG criteria (excluding pontine and spinal cord tumors), KPS/LPS≥60, ≥90 days from prior radiation, and adequate washout from prior anti-cancer therapy. The primary endpoint was overall response rate (ORR) by RANO-HGG criteria. Secondary endpoints included duration of response, time to response, progression-free survival (PFS), overall survival (OS), corticosteroid response rate, performance status response rate, and ORR by RANO-LGG criteria. Radiographic endpoints were assessed by dual-reader blinded independent central review. Data cutoff was May 31, 2021. RESULTS ORR was 20.0% (95%CI, 10.0–33.7) by RANO-HGG criteria. Median duration of response was 11.2 months (95%CI, 3.8–not reached) and median time to response was 8.3 months (range, 1.9–15.9). PFS at 6 months was 35.1% (95%CI, 21.2–49.3). The ORR was 26.0% (95%CI, 14.6–40.3) by RANO-LGG criteria. Fifteen patients (30.0%; 95%CI, 17.9–44.6) achieved an objective response by RANO-HGG and/or RANO-LGG criteria. Of 15 patients receiving ≥4 mg daily dexamethasone at baseline, 7 (46.7%; 95%CI, 21.3–73.4) achieved ≥50% confirmed reduction in dose. Of 34 patients with baseline KPS/LPS CONCLUSIONS ONC201 monotherapy exhibits durable and clinically meaningful efficacy in recurrent H3 K27M-mutant DMG.

BACKGROUND: Challenges around the design and investigation of cell culture models of adamantinomatous craniopharyngioma (ACP) have arisen from the cellular heterogeneity of these tumors, with populations that harbor disparate requirements in culture. Novel approaches to in vitro modeling of ACP are needed. METHODS: Intraoperatively collected tumor specimens were mechanically digested and plated under conditions tailored to the cell population of interest. ACP tumor-derived fibroblasts and epithelial cells were isolated using serum-containing and keratinocyte-specific media respectively. ACP-derived epithelial cells were immortalized via SV40 virus transfection and puromycin treatment for stable cell-line generation. Cell line validation included immunofluorescence with markers appropriate for the cell population of interest. RNA sequencing of cell lines was compared to ACP transcriptome reference data. Cell typing was conducted using short tandem repeat sequencing. RESULTS: ACP fibroblasts and ACP epithelial cells maintained spindle-like and cobblestone morphologies respectively, even after 4 passages. Immunofluorescence staining confirmed high levels of Vimentin expression in ACP-derived fibroblasts, and panCK and B-catenin in ACP-derived epithelial cells. Point mutation in exon 3 of the CTNNB1 gene was identified in ACP-derived epithelial cells. CONCLUSION: Initial limits related to cell line development in ACP may be addressed through the isolation and culture-specific ACP cell populations. This experience demonstrates the maintenance of validated markers of the cell populations of interest ex vivo. While preliminary, such cell lines offer promise as tools for the identification and study of potential therapeutic vulnerabilities in ACP.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a fatal brain tumor. Herein, we report on novel prognostic and predictive genomic biomarkers identified in PNOC003, a multi-center precision medicine trial for children and young adults diagnosed with DIPG. METHODS: Patients aged 3-25 years were enrolled on PNOC003 based on radiographic diagnosis of DIPG. Pre-treatment tumor biopsies were analyzed using tumor-normal whole-exome sequencing and mRNA-tumor sequencing to determine biology-informed, multi-agent therapy following radiation therapy (RT). Whole-genome sequencing was performed as an exploratory study aim. Genomic biomarkers were investigated to identify predictors of RT response and overall survival (OS) in patients with confirmed H3K27M-altered DIPG. Prognostic biomarkers were verified in a retrospective, H3K27M-altered diffuse midline glioma cohort (n=22) from the Children’s Brain Tumor Network (CBTN). RESULTS: Thirty patients enrolled on PNOC003 met molecular criteria for H3K27M-altered DIPG. TP53 was the most frequently altered driver gene (73%). Somatic alterations in PTEN>TP53>PDGFRA were independently associated with OS (P

INTRODUCTION: Autophagy is a highly conserved process by which intracellular components are degraded and recycled promoting cell survival. Preclinically, autophagy has been implicated as a resistance mechanism in BRAF-mutant glioma cells treated with MAPK-pathway inhibitors. HCQ, an oral autophagy inhibitor, has been evaluated preclinically and clinically to overcome resistance. METHODS: PBTC-055 (NCT04201457) is a phase I/II trial of HCQ combined with trametinib (BRAF-fusion or NF1-associated gliomas) or trametinib and dabrafenib (BRAFV600E gliomas) in patients < 30 years with progressive glioma. Prior treatment with RAF and/or MEK inhibitor with sub-optimal response (no response or response followed by progression on therapy) was required. Here, we present phase I data combining trametinib with HCQ utilizing a rolling-6 design. HCQ was administered at escalating dose levels (8, 15, or 20 mg/kg/day divided BID) in combination with standard pediatric trametinib dosing. All patients received prior MEK inhibitor therapy; 5/18 (28%) exhibited no response and 13/18 (72%) progressed on active therapy. RESULTS: Eighteen eligible/evaluable subjects were enrolled. Median age was 9.6 years (2.5-20.4 years); 10 were male. There were 2 dose-limiting toxicities (both grade 3 rash one each at DL1 and DL3). The highest dose level of HCQ (20 mg/kg/day divided BID) was declared the RP2D. Grade 3 adverse events possibly related to therapy included skin infection, rash, cardiac ejection fraction decrease, weight loss, and anorexia. There were no grade 4 or 5 attributable toxicities. Preliminarily, combination pharmacokinetic assessment revealed similar metabolism of trametinib to that reported as a single agent; HCQ demonstrated more rapid clearance compared to adults. Pharmacodynamic assessments are ongoing. CONCLUSIONS: The combination of trametinib and HCQ is safe with a RP2D of HCQ of 20 mg/kg/day divided BID. Currently, subjects are enrolling on the phase II portion evaluating the efficacy of this novel combination.

BACKGROUND: Molecular profiling of childhood CNS tumors is critical for diagnosis and clinical management, yet tissue access is restricted due to sensitive neuroanatomical locations. Moreover, CNS tumors including diffuse midline glioma (DMG) exhibit mutational heterogeneity and clonal evolution, which cannot be captured by upfront diagnostic biopsy alone. To address the lack of tumor visibility, and tprovide opportunity for longitudinal sampling, we validated and optimized a commercially available deep sequencing platform for analysis of circulating tumor DNA (TSO500ctDNATM). METHODS: In a proof-of-concept study, we defined the sensitivity, specificity, and clinical relevance of our novel ctDNA platform via analysis of paired tissue, CSF, and blood from children with DMG (n=10). Paired samples were assessed for concordance and sequencing results were compared to digital droplet PCR (ddPCR) detection of prognostic H3K27M mutation. RESULTS: DMG associated mutations in genes including H3-3A, H3C2, TP53, and ACVR1 were detected in ctDNA, including in CSF samples with low (

Background Pediatric low-grade gliomas (pLGGs) are the most common childhood brain tumor. Progression-free survival (PFS) is much lower than overall survival, emphasizing the need for alternative treatments. Sporadic (without neurofibromatosis type 1) optic pathway and hypothalamic gliomas (OPHGs) are often multiply recurrent and cause significant visual deficits. Recently, there has been a prioritization of functional outcomes. Methods We present results from children with recurrent/progressive OPHGs treated on a PBTC (Pediatric Brain Tumor Consortium) phase II trial evaluating efficacy of selumetinib (AZD6244, ARRY-142886) a MEK-1/2 inhibitor. Stratum 4 of PBTC-029 included patients with sporadic recurrent/progressive OPHGs treated with selumetinib at the recommended phase II dose (25mg/m2/dose BID) for a maximum of 26 courses. Results Twenty-five eligible and evaluable patients were enrolled with a median of 4 (1-11) previous therapies. Six of 25 (24%) had partial response, 14/25 (56%) had stable disease, and 5 (20%) had progressive disease while on treatment. The median treatment courses were 26 (2-26); 14/25 patients completed all 26 courses. Two-year PFS was 78 ± 8.5%. Nineteen of 25 patients were evaluable for visual acuity which improved in 4/19 patients (21%), was stable in 13/19 (68%), and worsened in 2/19 (11%). Five of 19 patients (26%) had improved visual fields and 14/19 (74%) were stable. The most common toxicities were grade 1/2 CPK elevation, anemia, diarrhea, headache, nausea/emesis, fatigue, AST and ALT increase, hypoalbuminemia, and rash. Conclusions Selumetinib was tolerable and led to responses and prolonged disease stability in children with recurrent/progressive OPHGs based upon radiographic response, PFS, and visual outcomes.

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=15) received autologous tumor antigen-associated T cells (TAAT) (up to 4x107/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 15/15 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 13/15 TAAT by a-IFN-γ ELISPOT. Plasma cytokine and proteomic analyses are ongoing but have demonstrated dynamic post-infusion immune cytokine and protein responses. Increases in the inflammatory and immune-stimulatory cytokines IL-1b, IL-6, IL-2 and IL-7 were observed post-infusion in most patients evaluated. Infusion-related increases in regulatory cytokines IL-10 and IL-13 were also observed in 4/7 patients. These results are consistent with an infusion-mediated immune response in vivo. Of 9 patients who have been tested thus far, 29/92 plasma proteins showed significant differences between dose levels 1 and 2, including increased IL-7 (p < 0.0004) and CD40L (p < 0.046) and reduced IL-4 (p < 0.0004). T cell receptor sequencing data on in vivo TAAT persistence is pending. In summary, TAAT have thus far been safe and elicit immune responses in vivo. Clinical and immunologic response assessments are ongoing.

Molecular-targeted therapy is an attractive therapeutic approach for childhood brain tumors. Unfortunately, with some notable exceptions, such treatment has not yet made a major impact on survival or for that matter quality-of-life for children with brain tumors. Limitations include the specificity of any single agent to inhibit the target, the presence of multiple genetic abnormalities within a tumor, the likely presence of escape mechanisms and the frequent use of molecular-targeted therapies in relatively biologically unselected patient populations. Despite these limitations, the MEK inhibitors and the BRAF V600E inhibitors have already demonstrated efficacy and are being compared to standard therapy in trials of treatment-naïve patients. There is also great enthusiasm for molecular-targeted therapies that target selective gene fusions. Given the plasticity of epigenetic changes, the targeting of epigenetic aberrations is also a promising avenue of therapy. Because molecular-targeted therapies frequently target genes and pathways that are critical in normal brain development, the acute, subacute long-term sequelae of molecular-targeted therapies need to be carefully monitored.

Background: BRAFV600E mutation is present in a subset of pediatric brain tumors. Vemurafenib is an oral, selective ATP-competitive inhibitor of BRAFV600E kinase. The goal of this multi-center study conducted through the Pacific Pediatric Neuro-Oncology Consortium (PNOC) was to determine the recommended phase 2 dose (RP2D) and dose limiting toxicities (DLTs) in children < 18 years with recurrent or progressive BRAFV600E mutant brain tumors. Results: Nineteen eligible patients were enrolled. Eleven patients had received three or more prior therapies. Data reported are from the start of treatment for the first patient (April 30 2014) through August 31 2019. The RP2D was defined as 550 mg/m2 twice daily after DLT criteria adjustment for rash. Related grade ≥ 3 adverse events included secondary keratoacanthoma (n = 1); rash (n =16); and fever (n = 5). Subjects received a median of 23 cycles (range 3-63). Four patients remain on treatment. Centrally reviewed best radiographic responses included 1 complete response, 5 partial responses, and 13 stable disease. The steady-state area under the curve (AUC0-∞median) was 604 mg*h/L (range 329-1052). Methods: Vemurafenib was given starting at 550 mg/m2, twice daily which corresponds to the adult RP2D. Adverse events were graded using the NIH Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Central imaging review was performed. Pharmacokinetic sampling was performed. Conclusions: Vemurafenib has promising anti-tumor activity in recurrent BRAF V600E-positive brain tumors with manageable toxicity. A phase 2 study is ongoing (NCT01748149).

Deep learning (DL) is a widely applied mathematical modeling technique. Classically, DL models utilize large volumes of training data, which are not available in many healthcare contexts. For patients with brain tumors, non-invasive diagnosis would represent a substantial clinical advance, potentially sparing patients from the risks associated with surgical intervention on the brain. Such an approach will depend upon highly accurate models built using the limited datasets that are available. Herein, we present a novel genetic algorithm (GA) that identifies optimal architecture parameters using feature embeddings from state-of-the-art image classification networks to identify the pediatric brain tumor, adamantinomatous craniopharyngioma (ACP). We optimized classification models for preoperative Computed Tomography (CT), Magnetic Resonance Imaging (MRI), and combined CT and MRI datasets with demonstrated test accuracies of 85.3%, 83.3%, and 87.8%, respectively. Notably, our GA improved baseline model performance by up to 38%. This work advances DL and its applications within healthcare by identifying optimized networks in small-scale data contexts. The proposed system is easily implementable and scalable for non-invasive computer-aided diagnosis, even for uncommon diseases.

Adamantinomatous craniopharyngioma (ACP) is a biologically benign but clinically aggressive lesion that has a significant impact on quality of life. The incidence of the disease has a bimodal distribution, with peaks occurring in children and older adults. Our group previously published the results of a transcriptome analysis of pediatric ACPs that identified several genes that were consistently overexpressed relative to other pediatric brain tumors and normal tissue. We now present the results of a transcriptome analysis comparing pediatric to adult ACP to identify biological differences between these groups that may provide novel therapeutic insights or support the assertion that potential therapies identified through the study of pediatric ACP may also have a role in adult ACP. Using our compiled transcriptome dataset of 27 pediatric and 9 adult ACPs, obtained through the Advancing Treatment for Pediatric Craniopharyngioma Consortium, we interrogated potential age-related transcriptional differences using several rigorous mathematical analyses. These included: canonical differential expression analysis; divisive, agglomerative, and probabilistic based hierarchical clustering; information theory based characterizations; and the deep learning approach, HD Spot. Our work indicates that there is no therapeutically relevant difference in ACP gene expression based on age. As such, potential therapeutic targets identified in pediatric ACP are also likely to have relvance for adult patients.

Response criteria for paediatric high-grade glioma vary historically and across different cooperative groups. The Response Assessment in Neuro-Oncology working group developed response criteria for adult high-grade glioma, but these were not created to meet the unique challenges in children with the disease. The Response Assessment in Pediatric Neuro-Oncology (RAPNO) working group, consisting of an international panel of paediatric and adult neuro-oncologists, clinicians, radiologists, radiation oncologists, and neurosurgeons, was established to address issues and unique challenges in assessing response in children with CNS tumours. We established a subcommittee to develop response assessment criteria for paediatric high-grade glioma. Current practice and literature were reviewed to identify major challenges in assessing the response of paediatric high-grade gliomas to various treatments. For areas in which scientific investigation was scarce, consensus was reached through an iterative process. RAPNO response assessment recommendations include the use of MRI of the brain and the spine, assessment of clinical status, and the use of corticosteroids or antiangiogenics. Imaging standards for brain and spine are defined. Compared with the recommendations for the management of adult high-grade glioma, for paediatrics there is inclusion of diffusion-weighted imaging and a higher reliance on T2-weighted fluid-attenuated inversion recovery. Consensus recommendations and response definitions have been established and, similar to other RAPNO recommendations, prospective validation in clinical trials is warranted.

This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly diagnosed patients (

METHODS Through the Advancing Treatment for Pediatric Craniopharyngioma (ATPC) consortium we accumulated preoperative MRIs and tumor RNA for 50 unique ACP patients. MRIs were assessed quantitatively for 28 different features and analyzed using Multiple Factor Analysis (MFA) and optimal clustering was determined via maximization of Bayesian Information Criterion (BIC). Following bulk RNAseq, differential expression and pathway enrichment were performed using standard methodologies (i.e., DESeq2 and GSEA). RESULTS MRI features were well represented in the first 3 dimensions of MFA (variance explained=67.32%); specifically tumor/cyst size, ventricular size, and cyst fluid diffusivity. Using this three-way axis, we identified 3 patient subgroups. Transcriptional differences between these subgroups indicated one group was enriched for DNA damage response and MYC related pathways, one group enriched for SHH, and one group enriched for WNT/β-catenin and EMT-related pathways. CONCLUSION This preliminary work suggests that there may be unique gene expression variants within ACP, which may be identified preoperatively using easily quantifiable MRI parameters. These radiogenomic signatures could provide prognostic information and/or guidance in the selection of antitumor therapies for children with ACP.

INTRODUCTION The role of diagnostic biopsy in diffuse intrinsic pontine glioma (DIPG) remains in question. Distinguishing radiographically between DIPG and other pontine tumors with more favorable prognosis and different therapy is critically important. METHODS Cases submitted to the International DIPG registry with histopathologic data were analyzed. Central imaging review was performed by two neuro-radiologists; all cases with imaging features or histopathology suggestive of alternative diagnoses were re-reviewed. Imaging features suggestive of alternative diagnoses included non-pontine origin, RESULTS Among 297 patients with pathology from biopsy and/or autopsy available, 27 (9%) had histologic diagnoses not consistent with DIPG, commonly embryonal tumors (n=9) and pilocytic astrocytomas (n=11). 163 patients had diagnostic MRI available for central neuroimaging review. Among 81 patients classified as characteristic of DIPG, 80 (99%) had histopathology consistent with DIPG (diffuse midline glioma, H3K27M-mutant, glioblastoma, anaplastic astrocytoma, diffuse astrocytoma). Among 63 patients classified as likely DIPG, but with unusual imaging features, 59 (94%) had histopathology consistent with DIPG. 19 patients had imaging features suggestive of another diagnosis, including 13 with non-pontine tumor origin; the remaining 6 all had histopathology not consistent with DIPG. Association between central imaging review and histopathology was significant (p CONCLUSIONS The important role and accuracy of central neuroimaging review in diagnosing or excluding DIPG is demonstrated. In patients with pontine tumors for which DIPG is felt unlikely radiographically, biopsy may be considered to guide diagnosis and treatment.

H3 K27M-mutant diffuse midline gliomas (DMG) have a dismal prognosis. We report an integrated analysis for ONC201, a DRD2 antagonist and ClpP agonist, in patients with recurrent H3 K27M DMG administered as monotherapy in 3 clinical studies (NCT03295396, n=20; NCT02525692, n=6; or expanded access, n=4) by independent, central radiology review among 30 patients. At baseline, patients had measurable contrast-enhancing disease by RANO criteria, KPS>60, and were >90 days from prior radiation. Patients with primary lesions that involved the pons or spinal cord or had evidence of leptomeningeal or cerebrospinal fluid dissemination were excluded. ONC201 was orally administered at 625 mg (scaled by body weight for patients < 18 years old) weekly in 29 patients and every 3 weeks in 1 patient. Median age was 31 years (range 8–70; two patients < 18). There were 16 women and 14 men. Tumors were predominantly thalamic (73%), with other locations including the cerebellum (5%), brainstem (non-pontine) (3%), basal ganglia (3%), and midbrain (3%). Median time from prior radiation was 7.5 months. The most frequent drug-related adverse events were low grade nausea (10%) and fatigue (10%). Nine patients (30%) had >50% regression of T1 contrast enhancement and 11 (36.7%) patients had regression of T2/FLAIR. Objective response rate and progression-free survival by RANO criteria, as well overall survival will be reported. Among 8 patients with sustained radiographic regressions, 6 were tapered off dexamethasone and 4 had improvement in KPS. Five patients (16.7%) remain on treatment with a median of 9.5 months (range 7.3–12). In conclusion, single agent ONC201 is well tolerated and clinically active in recurrent H3 K27M DMG patients.

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

BACKGROUND: Pediatric low-grade glioma (pLGG) is the most common CNS tumor of childhood. Progression-free survival (PFS) is much lower than overall survival emphasizing the need for alternative treatments. In addition, many children suffer functional morbidities such as visual and motor disturbances. Recently, there has been an appropriate prioritization of functional outcomes in children with pLGG. METHODS: We present the results of a PBTC phase II trial evaluating selumetinib, (AZD6244, ARRY-142886) a MEK-1/2 inhibitor, in children with recurrent/progressive pLGG on 2 strata, including visual outcomes. RESULTS: Stratum 3 enrolled Neurofibromatosis type 1 (NF1)-associated pLGG. Ten of 25 (40%) eligible patients had partial response (PR), 14/25 (56%) had stable disease (SD) and 1/25 (4%) had progressive disease (PD); 2-year PFS was 96+4%. Ten patients with optic pathway glioma (OPG) were evaluable for visual acuity (VA) at baseline and 1 year. VA improved in 2/10 patients (20%) and was stable in 8/10 (80%). One patient (10%) had improvement in visual fields (VF) and 9 patients (90%) had stable VF. Stratum 4 included patients with non-NF1-associated recurrent/progressive hypothalamic and OPG. Five of 25 (20%) eligible patients had PR, 16/25 (64%) had SD and 4 (16%) had PD; 2-year PFS was 78+8.5%. Nineteen of 25 patients were evaluable for VA. VA improved in 4/19 patients (21%), was stable in 13/19 (68%) and worsened in 2/19 (11%). Five patients (26%) had improved VF and 14 (74%) had stable VF. The most common toxicities included grade 1/2 CPK elevation, diarrhea, hypoalbuminemia and rash. Rare grade 3 toxicities included elevated CPK, rash and paronychia. CONCLUSIONS: Selumetinib was tolerable and effective in treating children with NF1-associated and sporadic recurrent/progressive hypothalamic and OPG based upon radiographic response and PFS. Twenty-seven of 29 (93%) evaluable patients had stable or improved vision based on VA and VF testing.

RATIONALE: A major factor contributing to the expanding knowledge of CNS tumors, specifically DMG biology is the selfless gift from patients and their families of postmortem tumor tissue. To facilitate this, we developed a postmortem donation program for collection of tissue and biospecimens for accelerating biology studies of end stage disease for pediatric CNS tumors. METHODS: Our postmortem program entails coordinating tissue donations, compiling clinical data, processing of whole brain, spinal cord, biofluids, and germline controls. Through our protocol, primary and disseminated tumor specimens are designated for molecular characterization and for generation of a matched preclinical model. RESULTS: During the past eight years, we coordinated and accrued over 50 autopsy cases including diffuse midline gliomas, ATRT, ETANTR, CPC from 20 institutions across North America and the United Kingdom. We report that approaching a family at the time of progression has provided the most successful outcome for consent to our program. Patient age range was from 11months to 20 years old. The median time from postmortem donation to tissue processing range was 13 hours with the earliest time point being 4 hours and the longest being 96 hours. All collected specimens were successfully processed and assessed for DNA/RNA integrity using bioanalyzer, attempted primary neurosphere cultures and cryopreserved for future molecular studies. PDX generation from fresh tissue proved more successful compared to primary neurospheres and post mortem time did not seem to have an effect. We have successfully generated models that represent molecular subgroups of DMGs such as H3.3/ H3.1- K27M and H3 WT. All generated preclinical models were validated by STR analysis and immunohistochemical assays using human mitochondrial antibodies. IMPACT: Postmortem tissue donations serve as an invaluable source for access to end-stage disease biology, generation of PDX models, studying patterns of tumor migration, testing combination therapy and preclinical drug testing.

BACKGROUND: Pediatric low-grade glioma (pLGG) is the most common central nervous system tumor of childhood. Although overall survival is very good, many children suffer from multiple progressions and functional morbidities. There is no one universally accepted therapy for children with recurrent disease, however, standard cytotoxic chemotherapies are often utilized by most practitioners. The Pediatric Brain Tumor Consortium conducted a multi-institutional phase II study evaluating selumetinib (AZD6244, ARRY-142886), a MAP/ERK Kinase I/II inhibitor, in patients with recurrent, refractory or progressive pLGG assigned to numerous strata. The aim of the study was to evaluate the efficacy of selumetinib in these patients. METHODS: Eligibility required age 3–21 y/o, a Lansky or Karnofsky performance score greater than 60 and the presence of recurrent, refractory or progressive pLGG after at least one standard therapy. Stratum 1 included children with World Health Organization (WHO) grade I pilocytic astrocytoma (PA) harboring either one of the two most common BRAF aberrations (KIAA1549-BRAF fusion or the BRAF(V600E) mutation). Stratum 3 included children with any neurofibromatosis type 1 (NF1)-associated pLGG (WHO grades I and II). Selumetinib was provided as capsules given orally at the recommended phase II dose of 25 mg/m(2) twice daily. The primary endpoint was stratum-specific objective response rate assessd by the local site and sustained for at least 8 weeks. All responses were reviewed centrally and statistical analyses were done as per protocol. Although the trial (NCT01089101) is still ongoing in other strata, enrollment and planned follow-up is compete on both strata 1 and 3. FINDINGS: Between July 25, 2013, and June 12, 2015, 25 eligible and evaluable children were accrued to stratum 1, and between August 28, 2013, and June 25, 2015, 25 eligible and evaluable children were accrued to stratum 3. On stratum 1, 9/25 (36%) patients achieved a partial response (PR). The median follow-up for the 11 patients who have not yet experienced an event is 36.4 months (4.4–50.5; IQR=23.9). On stratum 3, 10/25 (40%) patients achieved a PR with a median follow-up of 48.6 months (8.6–59.1; IQR=12.2) for the 17 subjects without progressions. All patients evaluable for visual acuity had improved or stable vision. The most common attributable toxicities on both strata were grade 1 and 2 CPK elevation, hypoalbuminemia, dyspnea, rash, duodenal ulcer, anemia, dry skin, fatigue and diarrhea. Rare grade 3 toxicities included elevated CPK (n=5), maculopapular rash (n=5), neutropenia (n=3), nausea (n=3), paronychia (n=3), acneiform rash (n=2), diarrhea (n=2), elevated ALT (n=1), decreased ejection fraction (n=1), gastric hemorrhage (n=1), headache (n=1), skin infection (n=1), tooth infection (n=1) and weight gain (n=1). There was only one grade 4 toxicity, lymphopenia. There were no treatment-realted deaths. Patient reported outcomes and quality of life assessments were not part of the current study. INTERPRETATION: Selumetinib is active against recurrent, refractory or progressive PA harboring common BRAF aberrations and NF1-associated pLGG. To our knowledge, this is one of the first prospectively tested and successful molecularly-targeted agents in pLGG. These data not only provide an alternative to standard chemotherapy for these subgroups of patients, but this success has led to an interest in exploring efficacy in patients as a first-line therapy. In fact, these data have directly led to the development of two Children’s Oncology Group phase III studies in newly diagnosed pLGG patients both with and without NF1 comparing standard chemotherapy to selumetinib. The current trial was funded by a National Cancer Institute (NCI) Cancer Therapy Evaluation Program (CTEP) PBTC U01 Grant: 2UM1CA081457 (UM1) and by the American Lebanese Syrian Associated Charities.

Anaplastic astrocytomas are aggressive glial cancers that present poor prognosis and high recurrence. Heterozygous IDH1 R132H mutations are common in adolescent and young adult anaplastic astrocytomas. In a majority of cases, the IDH1 R132H mutation is unique to the tumor, although rare cases of anaplastic astrocytoma have been described in patients with mosaic IDH1 mutations (Ollier disease or Maffucci syndrome). Here, we present two siblings with IDH1 R132H mutant high grade astrocytomas diagnosed at 14 and 26 years of age. Analysis of IDHR132H mutations in the siblings' tumors and non-neoplastic tissues, including healthy regions of the brain, cheek cells, and primary teeth indicate mosaicism of IDHR132H. Whole exome sequencing of the tumor tissue did not reveal any other common mutations between the two siblings. This study demonstrates the first example of IDH1 R132H mosaicism, acquired during early development, that provides an alternative mechanism of cancer predisposition.

Purpose: Pediatric diffuse midline glioma (DMG) are highly malignant tumors with poor clinical outcomes. Over 70% of patients with DMG harbor the histone 3 p.K27M (H3K27M) mutation, which correlates with a poorer clinical outcome, and is also used as a criterion for enrollment in clinical trials. Because complete surgical resection of DMG is not an option, biopsy at presentation is feasible, but rebiopsy at time of progression is rare. While imaging and clinical-based disease monitoring is the standard of care, molecular-based longitudinal characterization of these tumors is almost nonexistent. To overcome these hurdles, we examined whether liquid biopsy allows measurement of disease response to precision therapy. Experimental Design: We established a sensitive and specific methodology that detects major driver mutations associated with pediatric DMGs using droplet digital PCR (n = 48 subjects, n = 110 specimens). Quantification of circulating tumor DNA (ctDNA) for H3K27M was used for longitudinal assessment of disease response compared with centrally reviewed MRI data. Results: H3K27M was identified in cerebrospinal fluid (CSF) and plasma in 88% of patients with DMG, with CSF being the most enriched for ctDNA. We demonstrated the feasibility of multiplexing for detection of H3K27M, and additional driver mutations in patient's tumor and matched CSF, maximizing the utility of a single source of liquid biome. A significant decrease in H3K27M plasma ctDNA agreed with MRI assessment of tumor response to radiotherapy in 83% (10/12) of patients. Conclusions: Our liquid biopsy approach provides a molecularly based tool for tumor characterization, and is the first to indicate clinical utility of ctDNA for longitudinal surveillance of DMGs.

BACKGROUND Abnormal p53 function commonly defines high-risk CNS tumors, but functional restoration has eluded investigators. SGT-53 is a targeted nanomedicine encapsulating a plasmid DNA encoding wild-type human p53 with a transferrin receptor-targeting scFv on the nanocomplex surface resulting in efficient delivery across the BBB and robust tumor binding/uptake. Data generated by collaborators demonstrated synergy with irradiation and chemotherapy. REPORT: Two children with recurrent CNS malignancies have been treated with SGT-53, each receiving greater than 50 infusions in combination with irradiation and chemotherapy with no grade 3/4 AEs positively attributed to SGT-53. The first was an 11yo male with recurrent disseminated p53+ SHH medulloblastoma, with bulky intracranial/thoracolumbar disease. He received irradiation followed by biweekly doses of SGT-53 and temozolomide, bevacizumab and irinotecan given one week out of four. This patient exhibited a complete response of all disease on his first follow-up scan 8 weeks after therapy initiation and remained in remission for 8 months. The second patient was a 3yo male with disseminated recurrent choroid plexus carcinoma. He received CSI with SGT-53, followed by SGT-53, temozolomide and irinotecan as described above, again with no related grade 3/4 AEs. He experienced a partial response to all sites of disease and completed therapy after six months, progressing 7.8 months after initiating treatment. CONCLUSIONS SGT-53 was well tolerated in two heavily-pretreated patients despite aggressive combinatorial strategies. The majority of the AEs experienced were mild and manageable. Each patient had significant responses, suggesting that SGT-53 should be evaluated in a clinical trial for similar patients.

The PBTC conducted a re-treatment study (NCT01089101) evaluating selumetinib (AZD6244, ARRY-142886), a MEK I/II inhibitor, in children with recurrent/progressive pLGG. Eligible patients must have previously enrolled on PBTC-029 or PBTC-029B and progressed after coming off treatment with selumetinib. Patients must have maintained stable disease (SD) for ≥12 courses or had a sustained radiographic response (partial or complete) during their first exposure to selumetinib. Thirty-five eligible patients (median age: 13.11 years [range 7.96–25.33]) were enrolled, 57% of whom had optic pathway or hypothalamic target lesions. At the time of submission, median duration of treatment was 18 courses (range 2–48) and 21 subjects remained on therapy. Best responses reported to date are 6/35 (17%) partial response, 22/35 (63%) SD and 7/35 (20%) progressive disease with a 2-year progression-free survival of 75.7 + 8.3%, which met the design parameters for success. The most common attributable toxicities were grade 1 diarrhea, elevated AST, hypoalbuminemia, elevated CPK, maculo-papular rash, fatigue, paronychia, ALT elevation, acneiform rash and grade 2 CPK elevation. Rare grade 3 toxicities included CPK elevation (3), lymphopenia (2), paronychia (2) and ALT elevation (2). There was only one grade 4 CPK elevation. Five patients (14%) required dose reductions due to toxicity. There does not appear to be a notable difference in toxicities observed during initial selumetinib therapy versus re-treatment. In pLGG that has recurred/progressed following treatment with selumetinib, re-treatment with selumetinib appears to be effective with 80% of patients again achieving response or prolonged stable disease. Long-term follow-up is ongoing.

Recent improved understanding of the molecular markers of high grade glioma has shifted the approach to these aggressive CNS tumors to increasingly use molecularly guided targeted therapies. Treatment of patients with BRAFV600E mutant high grade gliomas with BRAF inhibitors has shown efficacy, however the impact of concomitant H3K27M mutation is unknown. ONC201 targets dopamine receptor D2 (DRD2), which is shown to be broadly overexpressed in the thalamus as well as multiple tumor types; its antagonism has demonstrated anti-tumor efficacy and immunomodulatory properties in preclinical studies. ONC201 has also demonstrated clinical efficacy in patients with H3K27M mutant gliomas. We present the case of a 9-year-old male with a right thalamic H3.3K27M mutant diffuse midline glioma with a concomitant BRAFV600E mutation with an ongoing partial response to ONC201 treatment. The patient was diagnosed in May 2018. He underwent biopsy, followed by standard focal proton radiation therapy (54Gy) and subsequent treatment with dasatinib, bevacizumab and everolimus over the course of five months. After continued radiographic progression on serial imaging, in April 2019 he started ONC201 375mg orally once per week through an expanded access trial. He has tolerated the medication well with grade 1 nausea and fatigue. Over the next nine months, he demonstrated clinical and radiographic improvement with modest increased use of his left side and MRIs showing progressive decrease in size of the thalamic lesion with a 70 % decrease in the target lesion (measuring 53x62mm prior to treatment, decreased to 38x26mm in January 2020).