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Central nervous system (CNS) tumors are the leading cause of cancer-related death in children. Typical therapy for CNS tumors in children involves a combination of surgery, radiation, and chemotherapy. While upfront therapy is effective for many high-grade tumors, therapy at the time of relapse remains limited. Furthermore, for diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), there are currently no curative therapies. Chimeric antigen receptor T (CAR T) cell therapy is a promising novel treatment avenue for these tumors. Here, we review the preclinical evidence for CAR T cell use in pediatric brain tumors, the preliminary clinical experience of CNS CAR T cell trials, toxicity associated with systemic and locoregional CAR T cell therapy for CNS tumors, challenges in disease response evaluation with CAR T cell therapy, and the knowledge gained from correlative biologic studies from these trials in the pediatric and young adult population., Competing Interests: Declarations Competing interests Dr. Vitanza is chair of the Scientific Board for Brain Child Bio. Otherwise, the authors have no financial or nonfinancial interests that are directly or indirectly related to the work submitted for publication., (© 2024. The Author(s).)

Purpose: B7-H3 is an immunoregulatory protein overexpressed by many pediatric solid tumors with limited expression on critical organs, making it an attractive immunotherapy target. We present a first-in-human phase I clinical trial systemically administered B7-H3 chimeric antigen receptor (CAR) T cells for young patients with relapsed or refractory solid tumors., Patients and Methods: Patients were enrolled onto a phase I trial to examine the safety of B7-H3-specific CARs at various dose levels (DLs) using a standard 3 + 3 dose escalation design., Results: Sixteen patients (range, 11-24 years; median, 18.5 years) were enrolled, and nine were treated at DL1 (0.5 × 10 6 CAR T cells/kg; n = 3) or DL2 (1 × 10 6 CAR T cells/kg; n = 6). There were no first infusion dose-limiting toxicities. Maximum first-infusion circulating CAR T cells detected in the peripheral blood were 4.98 cells/μL (range, 0-4.98 cells/μL) with detection of CAR T cells colocalizing with tumor cells at the site of metastatic disease in one patient. Patients were eligible for subsequent infusions. An objective partial response by PERCIST criteria was observed 28 days after a second CAR T cell infusion in a patient who did not have an objective response after the first infusion. The second infusion demonstrated marked enhancement of CAR T cell expansion to 1,590 cells/μL and was accompanied by cytokine release syndrome and dose-limiting transaminitis. Detailed peripheral blood cytokine profiling revealed elevated IL-21 levels preinfusion 2 compared with infusion 1., Conclusion: B7-H3 CAR T cells are tolerable and demonstrate limited antitumor activity without acute on-target, off-tumor toxicity. High levels of CAR T cell expansion may be necessary to achieve objective responses, but undefined host and tumor microenvironment factors appear to be critical (ClinicalTrials.gov identifier: NCT04483778).

While multiple factors impact disease, artificial intelligence (AI) studies in medicine often use small, non-diverse patient cohorts due to data sharing and privacy issues. Federated learning (FL) has emerged as a solution, enabling training across hospitals without direct data sharing. Here, we present FL-PedBrain, an FL platform for pediatric posterior fossa brain tumors, and evaluate its performance on a diverse, realistic, multi-center cohort. Pediatric brain tumors were targeted due to the scarcity of such datasets, even in tertiary care hospitals. Our platform orchestrates federated training for joint tumor classification and segmentation across 19 international sites. FL-PedBrain exhibits less than a 1.5% decrease in classification and a 3% reduction in segmentation performance compared to centralized data training. FL boosts segmentation performance by 20 to 30% on three external, out-of-network sites. Finally, we explore the sources of data heterogeneity and examine FL robustness in real-world scenarios with data imbalances., (© 2024. The Author(s).)

Background: A major obstacle in translating the therapeutic potential of chimeric antigen receptor (CAR) T cells to children with central nervous system (CNS) tumors is the blood-brain barrier. To overcome this limitation, preclinical and clinical studies have supported the use of repeated, locoregional intracranial CAR T-cell delivery. However, there is limited literature available describing the process for the involvement of an investigational drug service (IDS) pharmacy, particularly in the setting of a children's hospital with outpatient dosing for CNS tumors., Objectives: To describe Seattle Children's Hospital's experience in clinically producing CAR T cells and the implementation of IDS pharmacy practices used to deliver more than 300 intracranial CAR T-cell doses to children, as well as to share how we refined the processing techniques from CAR T-cell generation to the thawing of fractionated doses for intracranial delivery., Methods: Autologous CD4+ and CD8+ T cells were collected and transduced to express HER2, EGFR, or B7-H3-specific CAR T cells. Cryopreserved CAR T cells were thawed by the IDS pharmacy before intracranial delivery to patients with recurrent/refractory CNS tumors or with diffuse intrinsic pontine glioma/diffuse midline glioma., Results: The use of a thaw-and-dilute procedure for cryopreserved individual CAR T-cell doses provides reliable viability and is more efficient than typical thaw-and-wash protocols. Cell viability with the thaw-and-dilute protocol was approximately 75% and was always within 10% of the viability assessed at cryopreservation. Cell viability was preserved through 6 hours after thawing, which exceeded the 1-hour time frame from thawing to infusion., Conclusion: As the field of adoptive immunotherapy grows and continues to bring hope to patients with fatal CNS malignancies, it is critical to focus on improving the preparatory steps for CAR T-cell delivery., Competing Interests: Author Disclosure Statement Dr Vitanza serves as the scientific advisory board chair for BrainChild Bio, Inc. Dr Foster is a consultant for Teladoc and has received honoraria/royalties from Tmunity; Dr Gust is a consultant at Johnson & Johnson; Dr Gardner has received royalties from Juno Therapeutics and has received research support from 2seventy bio; Dr Jensen is the Chief Scientific Officer of BrainChild Bio and has received research and clinical trial support from Umoja Biopharma and 2seventy bio. Drs Vitanza, Foster, Gardner, and Jensen are inventors on issued and pending patents related to CAR T-cell therapies. Dr Choe, Mr Brown, Mr Beebe, Dr Kong, Dr Rogers, Dr Jacob, Ms Mano, Ms Abuan, Ms Mgebroff, Ms Lindgren, Dr Gustafson, Dr Wilson, Ms Noll, Dr Ronsley, Dr Crotty, Dr Leary, and Dr Park have no conflicts of interest to report.

Background: Liquid biopsy assays that detect cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) are a promising tool for disease monitoring in pediatric patients with primary central nervous system (CNS) tumors. As a compliment to tissue-derived molecular analyses, CSF liquid biopsy has the potential to transform risk stratification, prognostication, and precision medicine approaches., Methods: In this pilot study, we evaluated a clinical pipeline to determine feasibility and sensitivity of low-pass whole genome sequencing (LP-WGS) of CSF-derived cfDNA from patients with CNS embryonal tumors. Thirty-two longitudinal CSF samples collected from 17 patients with molecularly characterized medulloblastoma (12), embryonal tumor with multilayered rosettes (2), CNS embryonal tumor, not elsewhere classified (NEC) (2), and atypical teratoid/rhabdoid tumor (1) were analyzed., Results: Adequate CSF-derived cfDNA for LP-WGS analysis was obtained in 94% of samples (30/32). Copy number variants compatible with neoplasia were detected in 90% (27/30) and included key alterations, such as isodicentric ch17, monosomy 6, and MYCN amplification, among others. Compared to tissue specimens, LP-WGS detected additional aberrations in CSF not previously identified in corresponding primary tumor specimens, suggesting a more comprehensive profile of tumor heterogeneity or evolution of cfDNA profiles over time. Among the 12 CSF samples obtained at initial staging, only 2 (17%) were cytologically positive, compared to 11 (92%) that were copy number positive by LP-WGS., Conclusions: LP-WGS of CSF-derived cfDNA is feasible using a clinical platform, with greater sensitivity for tumor detection compared to conventional CSF cytologic analysis at initial staging. Large prospective studies are needed to further evaluate LP-WGS as a predictive biomarker., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: This study evaluated the safety and pharmacokinetics (PK) of oral ONC201 administered twice-weekly on consecutive days (D1D2) in pediatric patients with newly diagnosed DIPG and/or recurrent/refractory H3 K27M glioma., Methods: This phase 1 dose-escalation and expansion study included pediatric patients with H3 K27M-mutant glioma and/or DIPG following ≥1 line of therapy (NCT03416530). ONC201 was administered D1D2 at 3 dose levels (DLs; -1, 1, and 2). The actual administered dose within DLs was dependent on weight. Safety was assessed in all DLs; PK analysis was conducted in DL2. Patients receiving once-weekly ONC201 (D1) served as a PK comparator., Results: Twelve patients received D1D2 ONC201 (DL1, n = 3; DL1, n = 3; DL2, n = 6); no dose-limiting toxicities or grade ≥3 treatment-related adverse events occurred. PK analyses at DL2 (D1-250 mg, n = 3; D1-625 mg, n = 3; D1D2-250 mg, n = 2; D1D2-625 mg, n = 2) demonstrated variability in Cmax, AUC0-24, and AUC0-48, with comparable exposures across weight groups. No accumulation occurred with D1D2 dosing; the majority of ONC201 cleared before administration of the second dose. Cmax was variable between groups but did not appear to increase with D1D2 dosing. AUC0-48 was greater with D1D2 than once-weekly., Conclusions: ONC201 given D1D2 was well tolerated at all DLs and associated with greater AUC0-48., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Diffuse midline glioma (DMG) is a fatal pediatric cancer of the central nervous system (CNS). The location and infiltrative nature of DMG prevents surgical resection and the benefits of palliative radiotherapy are temporary; median overall survival (OS) is 9-11 months. The tumor immune microenvironment (TIME) is 'cold', and has a dominant immunosuppressive myeloid compartment with low levels of infiltrating lymphocytes and proinflammatory molecules. Because survival statistics have been stagnant for many decades, and therapies targeting the unique biology of DMG are urgently needed, this has prompted the clinical assessment of chimeric antigen receptor (CAR) T cell therapies in this setting. We highlight the current landscape of CAR T cell therapy for DMG, the role the TIME may play in the response, and strategies to overcome treatment obstacles., Competing Interests: Declaration of interests M.D.D. is a parent to a child lost to diffuse intrinsic pontine glioma (DIPG) and is the founder and a Director of the not-for-profit charity RUN DIPG Pty Ltd. The other authors declare no conflicts of interest., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with the development of hypermutant pediatric high-grade glioma, and confers a poor prognosis. While therapeutic histone deacetylase (HDAC) inhibition of diffuse intrinsic pontine glioma (DIPG) has been reported; here, we use a clinically relevant biopsy-derived hypermutant DIPG model (PBT-24FH) and a CRISPR-Cas9 induced genetic model to evaluate the efficacy of HDAC inhibition against hypermutant DIPG. We screened PBT-24FH cells for sensitivity to a panel of HDAC inhibitors (HDACis) in vitro, identifying two HDACis associated with low nanomolar IC50s, quisinostat (27 nM) and romidepsin (2 nM). In vivo, quisinostat proved more efficacious, inducing near-complete tumor regression in a PBT-24FH flank model. RNA sequencing revealed significant quisinostat-driven changes in gene expression, including upregulation of neural and pro-inflammatory genes. To validate the observed potency of quisinostat in vivo against additional hypermutant DIPG models, we tested quisinostat in genetically-induced mismatch repair (MMR)-deficient DIPG flank tumors, demonstrating that loss of MMR function increases sensitivity to quisinostat in vivo. Here, we establish the preclinical efficacy of quisinostat against hypermutant DIPG, supporting further investigation of epigenetic targeting of hypermutant pediatric cancers with the potential for clinical translation. These findings support further investigation of HDAC inhibitors against pontine high-grade gliomas, beyond only those with histone mutations, as well as against other hypermutant central nervous system tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Purpose of Review: Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies., Recent Findings: We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials., (© 2023. The Author(s).)

While immunotherapy for pediatric cancer has made great strides in recent decades, including the FDA approval of agents such as dinutuximab and tisgenlecleucel, these successes have rarely impacted children with pediatric central nervous system (CNS) tumors. As our understanding of the biological underpinnings of these tumors evolves, new immunotherapeutics are undergoing rapid clinical translation specifically designed for children with CNS tumors. Most recently, there have been notable clinical successes with oncolytic viruses, vaccines, adoptive cellular therapy, and immune checkpoint inhibition. In this article, the immunotherapy working group of the Pacific Pediatric Neuro-Oncology Consortium (PNOC) reviews the current and future state of immunotherapeutic CNS clinical trials with a focus on clinical trial development. Based on recent therapeutic trials, we discuss unique immunotherapy clinical trial challenges, including toxicity considerations, disease assessment, and correlative studies. Combinatorial strategies and future directions will be addressed. Through internationally collaborative efforts and consortia, we aim to direct this promising field of immuno-oncology to the next frontier of successful application against pediatric CNS tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992., Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib., (©2023 The Authors; Published by the American Association for Cancer Research.)

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

Cancer immunotherapies have unique toxicities. Establishment of grading scales and standardized grade-based treatment algorithms for toxicity syndromes can improve the safety of these treatments, as observed for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) in patients with B cell malignancies treated with chimeric antigen receptor (CAR) T cell therapy. We have observed a toxicity syndrome, distinct from CRS and ICANS, in patients treated with cell therapies for tumors in the central nervous system (CNS), which we term tumor inflammation-associated neurotoxicity (TIAN). Encompassing the concept of 'pseudoprogression,' but broader than inflammation-induced edema alone, TIAN is relevant not only to cellular therapies, but also to other immunotherapies for CNS tumors. To facilitate the safe administration of cell therapies for patients with CNS tumors, we define TIAN, propose a toxicity grading scale for TIAN syndrome and discuss the potential management of this entity, with the goal of standardizing both reporting and management., (© 2023. Springer Nature America, Inc.)

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors, raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation., Significance: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted., Methods: Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected., Results: Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial., Conclusions: H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: ONC201, a dopamine receptor D2 (DRD2) antagonist and caseinolytic protease P (ClpP) agonist, has induced durable tumor regressions in adults with recurrent H3 K27M-mutant glioma. We report results from the first phase I pediatric clinical trial of ONC201., Methods: This open-label, multi-center clinical trial (NCT03416530) of ONC201 for pediatric H3 K27M-mutant diffuse midline glioma (DMG) or diffuse intrinsic pontine glioma (DIPG) employed a dose-escalation and dose-expansion design. The primary endpoint was the recommended phase II dose (RP2D). A standard 3 + 3 dose escalation design was implemented. The target dose was the previously established adult RP2D (625 mg), scaled by body weight. Twenty-two pediatric patients with DMG/DIPG were treated following radiation; prior lines of systemic therapy in addition to radiation were permitted providing sufficient time had elapsed prior to study treatment., Results: The RP2D of orally administered ONC201 in this pediatric population was determined to be the adult RP2D (625 mg), scaled by body weight; no dose-limiting toxicities (DLT) occurred. The most frequent treatment-emergent Grade 1-2 AEs were headache, nausea, vomiting, dizziness and increase in alanine aminotransferase. Pharmacokinetics were determined following the first dose: T 1/2 , 8.4 h; T max , 2.1 h; C max , 2.3 µg/mL; AUC 0-tlast , 16.4 hµg/mL. Median duration of treatment was 20.6 weeks (range 5.1-129). Five (22.7%) patients, all of whom initiated ONC201 following radiation and prior to recurrence, were alive at 2 years from diagnosis., Conclusions: The adult 625 mg weekly RP2D of ONC201 scaled by body weight was well tolerated. Further investigation of ONC201 for DMG/DIPG is warranted., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs., Methods: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq., Results: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state., Conclusion: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065)., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Diffuse midline glioma (DMG), including those of the brainstem (diffuse intrinsic pontine glioma), are pediatric tumors of the central nervous system (CNS). Recognized as the most lethal of all childhood cancers, palliative radiotherapy remains the only proven treatment option, however, even for those that respond, survival is only temporarily extended. DMG harbor an immunologically "cold" tumor microenvironment (TME) with few infiltrating immune cells. The mechanisms underpinning the cold TME are not well understood. Low expression levels of immune checkpoint proteins, including PD-1, PD-L1, and CTLA-4, are recurring features of DMG and likely contribute to the lack of response to immune checkpoint inhibitors (ICIs). The unique epigenetic signatures (including stem cell-like methylation patterns), a low tumor mutational burden, and recurring somatic mutations (H3K27M, TP53, ACVR1, MYC, and PIK3CA), possibly play a role in the reduced efficacy of traditional immunotherapies. Therefore, to circumvent the lack of efficacy thus far seen for the use of ICIs, adoptive cell transfer (including chimeric antigen receptor T cells) and the use of oncolytic viruses, are currently being evaluated for the treatment of DMG. It remains an absolute imperative that we improve our understanding of DMG's intrinsic and TME features if patients are to realize the potential benefits offered by these sophisticated treatments. Herein, we summarize the limitations of immunotherapeutic approaches, highlight the emerging safety and clinical efficacy shown for sophisticated cell-based therapies, as well as the evolving knowledge underpinning the DMG-immune axis, to guide the development of immunotherapies that we hope will improve outcomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis., Methods: We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n = 24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI)., Results: Change in H3.3K27M VAF over time ("VAF delta") correlated with prolonged PFS in both CSF and plasma samples. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .085). VAF "spikes" (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression., Conclusion: Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background Radiogenomics of pediatric medulloblastoma (MB) offers an opportunity for MB risk stratification, which may aid therapeutic decision making, family counseling, and selection of patient groups suitable for targeted genetic analysis. Purpose To develop machine learning strategies that identify the four clinically significant MB molecular subgroups. Materials and Methods In this retrospective study, consecutive pediatric patients with newly diagnosed MB at MRI at 12 international pediatric sites between July 1997 and May 2020 were identified. There were 1800 features extracted from T2- and contrast-enhanced T1-weighted preoperative MRI scans. A two-stage sequential classifier was designed-one that first identifies non-wingless ( WNT ) and non-sonic hedgehog ( SHH ) MB and then differentiates therapeutically relevant WNT from SHH . Further, a classifier that distinguishes high-risk group 3 from group 4 MB was developed. An independent, binary subgroup analysis was conducted to uncover radiomics features unique to infantile versus childhood SHH subgroups. The best-performing models from six candidate classifiers were selected, and performance was measured on holdout test sets. CIs were obtained by bootstrapping the test sets for 2000 random samples. Model accuracy score was compared with the no-information rate using the Wald test. Results The study cohort comprised 263 patients (mean age ± SD at diagnosis, 87 months ± 60; 166 boys). A two-stage classifier outperformed a single-stage multiclass classifier. The combined, sequential classifier achieved a microaveraged F1 score of 88% and a binary F1 score of 95% specifically for WNT . A group 3 versus group 4 classifier achieved an area under the receiver operating characteristic curve of 98%. Of the Image Biomarker Standardization Initiative features, texture and first-order intensity features were most contributory across the molecular subgroups. Conclusion An MRI-based machine learning decision path allowed identification of the four clinically relevant molecular pediatric medulloblastoma subgroups. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Chaudhary and Bapuraj in this issue.

Background: The risk profile for posterior fossa ependymoma (EP) depends on surgical and molecular status [Group A (PFA) versus Group B (PFB)]. While subtotal tumor resection is known to confer worse prognosis, MRI-based EP risk-profiling is unexplored. We aimed to apply machine learning strategies to link MRI-based biomarkers of high-risk EP and also to distinguish PFA from PFB., Methods: We extracted 1800 quantitative features from presurgical T2-weighted (T2-MRI) and gadolinium-enhanced T1-weighted (T1-MRI) imaging of 157 EP patients. We implemented nested cross-validation to identify features for risk score calculations and apply a Cox model for survival analysis. We conducted additional feature selection for PFA versus PFB and examined performance across three candidate classifiers., Results: For all EP patients with GTR, we identified four T2-MRI-based features and stratified patients into high- and low-risk groups, with 5-year overall survival rates of 62% and 100%, respectively (P < .0001). Among presumed PFA patients with GTR, four T1-MRI and five T2-MRI features predicted divergence of high- and low-risk groups, with 5-year overall survival rates of 62.7% and 96.7%, respectively (P = .002). T1-MRI-based features showed the best performance distinguishing PFA from PFB with an AUC of 0.86., Conclusions: We present machine learning strategies to identify MRI phenotypes that distinguish PFA from PFB, as well as high- and low-risk PFA. We also describe quantitative image predictors of aggressive EP tumors that might assist risk-profiling after surgery. Future studies could examine translating radiomics as an adjunct to EP risk assessment when considering therapy strategies or trial candidacy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pineal anlage tumor is a rare pediatric tumor with clinical and histological features overlapping with pineoblastoma. Two patients with pineal anlage tumor, a 13-month-old female and an 11-month-old male, underwent subtotal resection, high-dose chemotherapy with autologous stem cell rescue, and radiation. Neither had tumor progression 50 months after diagnosis. The tumors underwent next-generation sequencing on a panel of 340 genes. Chromosomal copy gains and losses were present and differed between the tumors. No mutations or amplifications, including none specific to pineoblastoma, were identified., (© 2022 Wiley Periodicals LLC.)

Background and Purpose: Pediatric supratentorial tumors such as embryonal tumors, high-grade gliomas, and ependymomas are difficult to distinguish by histopathology and imaging because of overlapping features. We applied machine learning to uncover MR imaging-based radiomics phenotypes that can differentiate these tumor types., Materials and Methods: Our retrospective cohort of 231 patients from 7 participating institutions had 50 embryonal tumors, 127 high-grade gliomas, and 54 ependymomas. For each tumor volume, we extracted 900 Image Biomarker Standardization Initiative-based PyRadiomics features from T2-weighted and gadolinium-enhanced T1-weighted images. A reduced feature set was obtained by sparse regression analysis and was used as input for 6 candidate classifier models. Training and test sets were randomly allocated from the total cohort in a 75:25 ratio., Results: The final classifier model for embryonal tumor-versus-high-grade gliomas identified 23 features with an area under the curve of 0.98; the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 0.85, 0.91, 0.79, 0.94, and 0.89, respectively. The classifier for embryonal tumor-versus-ependymomas identified 4 features with an area under the curve of 0.82; the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 0.93, 0.69, 0.76, 0.90, and 0.81, respectively. The classifier for high-grade gliomas-versus-ependymomas identified 35 features with an area under the curve of 0.96; the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 0.82, 0.94, 0.82, 0.94, and 0.91, respectively., Conclusions: In this multi-institutional study, we identified distinct radiomic phenotypes that distinguish pediatric supratentorial tumors, high-grade gliomas, and ependymomas with high accuracy. Incorporation of this technique in diagnostic algorithms can improve diagnosis, risk stratification, and treatment planning., (© 2022 by American Journal of Neuroradiology.)

Diffuse midline glioma (DMG) is a deadly pediatric and adolescent central nervous system (CNS) tumor localized along the midline structures of the brain atop the spinal cord. With a median overall survival (OS) of just 9-11-months, DMG is characterized by global hypomethylation of histone H3 at lysine 27 (H3K27me3), driven by recurring somatic mutations in H3 genes including, HIST1H3B/C (H3.1K27M) or H3F3A (H3.3K27M), or through overexpression of EZHIP in patients harboring wildtype H3. The recent World Health Organization's 5th Classification of CNS Tumors now designates DMG as, 'H3 K27-altered', suggesting that global H3K27me3 hypomethylation is a ubiquitous feature of DMG and drives devastating transcriptional programs for which there are no treatments. H3-alterations co-segregate with various other somatic driver mutations, highlighting the high-level of intertumoral heterogeneity of DMG. Furthermore, DMG is also characterized by very high-level intratumoral diversity with tumors harboring multiple subclones within each primary tumor. Each subclone contains their own combinations of driver and passenger lesions that continually evolve, making precision-based medicine challenging to successful execute. Whilst the intertumoral heterogeneity of DMG has been extensively investigated, this is yet to translate to an increase in patient survival. Conversely, our understanding of the non-genomic factors that drive the rapid growth and fatal nature of DMG, including endogenous and exogenous microenvironmental influences, neurological cues, and the posttranscriptional and posttranslational architecture of DMG remains enigmatic or at best, immature. However, these factors are likely to play a significant role in the complex biological sequelae that drives the disease. Here we summarize the heterogeneity of DMG and emphasize how analysis of the posttranslational architecture may improve treatment paradigms. We describe factors that contribute to treatment response and disease progression, as well as highlight the potential for pharmaco-proteogenomics (i.e., the integration of genomics, proteomics and pharmacology) in the management of this uniformly fatal cancer., (© 2021. The Author(s).)

Background: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound-ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc., Methods: Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017-2020) was analyzed., Results: GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months ( P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not ( P = .012)., Conclusions: This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Extramedullary hematopoiesis (EMH) is hematopoiesis occurring outside of the bone marrow. It has been reported to develop in abdominal organs or lymph nodes after chemotherapy. Here, the authors describe a patient with a localized central nervous system embryonal tumor who, during intensive chemotherapy, developed dural nodules. Biopsy revealed these nodules to be EMH. Without a pathologic diagnosis, this may have been considered disease progression, altering the patient's treatment plan. This report intends to serve as a reminder that EMH should be included in the differential diagnosis of suspicious lesions and highlights the importance of their biopsy because of potential management implications., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Clinicians and machine classifiers reliably diagnose pilocytic astrocytoma (PA) on magnetic resonance imaging (MRI) but less accurately distinguish medulloblastoma (MB) from ependymoma (EP). One strategy is to first rule out the most identifiable diagnosis., Objective: To hypothesize a sequential machine-learning classifier could improve diagnostic performance by mimicking a clinician's strategy of excluding PA before distinguishing MB from EP., Methods: We extracted 1800 total Image Biomarker Standardization Initiative (IBSI)-based features from T2- and gadolinium-enhanced T1-weighted images in a multinational cohort of 274 MB, 156 PA, and 97 EP. We designed a 2-step sequential classifier - first ruling out PA, and next distinguishing MB from EP. For each step, we selected the best performing model from 6-candidate classifier using a reduced feature set, and measured performance on a holdout test set with the microaveraged F1 score., Results: Optimal diagnostic performance was achieved using 2 decision steps, each with its own distinct imaging features and classifier method. A 3-way logistic regression classifier first distinguished PA from non-PA, with T2 uniformity and T1 contrast as the most relevant IBSI features (F1 score 0.8809). A 2-way neural net classifier next distinguished MB from EP, with T2 sphericity and T1 flatness as most relevant (F1 score 0.9189). The combined, sequential classifier was with F1 score 0.9179., Conclusion: An MRI-based sequential machine-learning classifiers offer high-performance prediction of pediatric posterior fossa tumors across a large, multinational cohort. Optimization of this model with demographic, clinical, imaging, and molecular predictors could provide significant advantages for family counseling and surgical planning., (© Congress of Neurological Surgeons 2021.)

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for a global pandemic that can cause severe infections in children, especially those with comorbid conditions. Here, we report a case of a child with a newly diagnosed medulloblastoma, Fanconi Anemia, and SARS-CoV-2 infection. Through multidisciplinary care coordination and meticulous planning, we were able to safely initiate this patient's oncology care and implement a long-term model to address the patient's care. This approach could be replicated with any newly diagnosed pediatric patient that requires monitoring for signs of COVID-19 with concurrent oncology care., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background and Purpose: Atypical teratoid/rhabdoid tumors and medulloblastomas have similar imaging and histologic features but distinctly different outcomes. We hypothesized that they could be distinguished by MR imaging-based radiomic phenotypes., Materials and Methods: We retrospectively assembled T2-weighted and gadolinium-enhanced T1-weighted images of 48 posterior fossa atypical teratoid/rhabdoid tumors and 96 match-paired medulloblastomas from 7 institutions. Using a holdout test set, we measured the performance of 6 candidate classifier models using 6 imaging features derived by sparse regression of 900 T2WI and 900 T1WI Imaging Biomarker Standardization Initiative-based radiomics features., Results: From the originally extracted 1800 total Imaging Biomarker Standardization Initiative-based features, sparse regression consistently reduced the feature set to 1 from T1WI and 5 from T2WI. Among classifier models, logistic regression performed with the highest AUC of 0.86, with sensitivity, specificity, accuracy, and F1 scores of 0.80, 0.82, 0.81, and 0.85, respectively. The top 3 important Imaging Biomarker Standardization Initiative features, by decreasing order of relative contribution, included voxel intensity at the 90th percentile, inverse difference moment normalized, and kurtosis-all from T2WI., Conclusions: Six quantitative signatures of image intensity, texture, and morphology distinguish atypical teratoid/rhabdoid tumors from medulloblastomas with high prediction performance across different machine learning strategies. Use of this technique for preoperative diagnosis of atypical teratoid/rhabdoid tumors could significantly inform therapeutic strategies and patient care discussions., (© 2021 by American Journal of Neuroradiology.)

Inhibition of mTORC1 signaling has been shown to diminish growth of meningiomas and schwannomas in preclinical studies, and clinical data suggest that everolimus, an orally administered mTORC1 inhibitor, may slow tumor progression in a subset of patients with neurofibromatosis type 2 (NF2) with vestibular schwannoma. To assess the pharmacokinetics, pharmacodynamics, and potential mechanisms of treatment resistance, we performed a presurgical (phase 0) clinical trial of everolimus in patients undergoing elective surgery for vestibular schwannoma or meningiomas. Eligible patients with meningioma or vestibular schwannoma requiring tumor resection enrolled on study received everolimus 10 mg daily for 10 days immediately prior to surgery. Everolimus blood levels were determined immediately before and after surgery. Tumor samples were collected intraoperatively. Ten patients completed protocol therapy. Median pre- and postoperative blood levels of everolimus were found to be in a high therapeutic range (17.4 ng/mL and 9.4 ng/mL, respectively). Median tumor tissue drug concentration determined by mass spectrometry was 24.3 pg/mg (range, 9.2-169.2). We observed only partial inhibition of phospho-S6 in the treated tumors, indicating incomplete target inhibition compared with control tissues from untreated patients ( P = 0.025). Everolimus led to incomplete inhibition of mTORC1 and downstream signaling. These data may explain the limited antitumor effect of everolimus observed in clinical studies for patients with NF2 and will inform the design of future preclinical and clinical studies targeting mTORC1 in meningiomas and schwannomas., (©2021 American Association for Cancer Research.)

Background: Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis, with less than 2% surviving 5 years postdiagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified a few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically relevant DMG models to identify and validate other effective HDACi and their biomarkers of response., Methods: HDAC inhibitors were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically relevant radiation resistance. RNA sequencing was performed to define and compare drug efficacy and to map predictive biomarkers of response., Results: Quisinostat and romidepsin showed efficacy with low nanomolar half-maximal inhibitory concentration (IC50) values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all 3 drugs at similar biologically effective doses, such as overexpression of troponin T1 slow skeletal type (TNNT1) and downregulation of collagen type 20 alpha 1 chain (COL20A1), identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (P < 0.0001) inhibited in vivo tumor growth., Conclusions: Our data highlight the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain tumor-penetrant versions of potentially efficacious agents., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors. Presently, MRI is the mainstay of disease diagnosis and surveillance. We identify clinically significant computational features from MRI and create a prognostic machine learning model., Methods: We isolated tumor volumes of T1-post-contrast (T1) and T2-weighted (T2) MRIs from 177 treatment-naïve DIPG patients from an international cohort for model training and testing. The Quantitative Image Feature Pipeline and PyRadiomics was used for feature extraction. Ten-fold cross-validation of least absolute shrinkage and selection operator Cox regression selected optimal features to predict overall survival in the training dataset and tested in the independent testing dataset. We analyzed model performance using clinical variables (age at diagnosis and sex) only, radiomics only, and radiomics plus clinical variables., Results: All selected features were intensity and texture-based on the wavelet-filtered images (3 T1 gray-level co-occurrence matrix (GLCM) texture features, T2 GLCM texture feature, and T2 first-order mean). This multivariable Cox model demonstrated a concordance of 0.68 (95% CI: 0.61-0.74) in the training dataset, significantly outperforming the clinical-only model ( C = 0.57 [95% CI: 0.49-0.64]). Adding clinical features to radiomics slightly improved performance ( C = 0.70 [95% CI: 0.64-0.77]). The combined radiomics and clinical model was validated in the independent testing dataset ( C = 0.59 [95% CI: 0.51-0.67], Noether's test P = .02)., Conclusions: In this international study, we demonstrate the use of radiomic signatures to create a machine learning model for DIPG prognostication. Standardized, quantitative approaches that objectively measure DIPG changes, including computational MRI evaluation, could offer new approaches to assessing tumor phenotype and serve a future role for optimizing clinical trial eligibility and tumor surveillance., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal tumor of the brainstem, most commonly affecting young children. Due to its location, surgical resection is not achievable, but consideration of a biopsy has become standard practice at children's hospitals with the appropriate neurosurgical expertise. While the decision to obtain a biopsy should be directed by the presence of atypical radiographic features that call the diagnosis of DIPG into question or the requirement of biopsy tissue for clinical trial enrollment, once this precious tissue is available its use for research should be considered. The majority of DIPG and diffuse midline glioma, H3 K27M-mutant (DMG) models are autopsy-derived or genetically-engineered, each of which has limitations for translational studies, so the use of biopsy tissue for laboratory model development provides an opportunity to create unique model systems. Here, we present a detailed laboratory protocol for the generation of treatment-naïve biopsy-derived DIPG/DMG models., Competing Interests: Conflicts of Interest None.

Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. We show that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNAreplication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, we demonstrate that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. We propose that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis., Competing Interests: JS, MM, DJ, JH, HF, PP, CL, NV, JO, KA, SH No competing interests declared, (© 2020, Sarthy et al.)

Background and Purpose: Posterior fossa tumors are the most common pediatric brain tumors. MR imaging is key to tumor detection, diagnosis, and therapy guidance. We sought to develop an MR imaging-based deep learning model for posterior fossa tumor detection and tumor pathology classification., Materials and Methods: The study cohort comprised 617 children (median age, 92 months; 56% males) from 5 pediatric institutions with posterior fossa tumors: diffuse midline glioma of the pons ( n = 122), medulloblastoma ( n = 272), pilocytic astrocytoma ( n = 135), and ependymoma ( n = 88). There were 199 controls. Tumor histology served as ground truth except for diffuse midline glioma of the pons, which was primarily diagnosed by MR imaging. A modified ResNeXt-50-32x4d architecture served as the backbone for a multitask classifier model, using T2-weighted MRIs as input to detect the presence of tumor and predict tumor class. Deep learning model performance was compared against that of 4 radiologists., Results: Model tumor detection accuracy exceeded an AUROC of 0.99 and was similar to that of 4 radiologists. Model tumor classification accuracy was 92% with an F 1 score of 0.80. The model was most accurate at predicting diffuse midline glioma of the pons, followed by pilocytic astrocytoma and medulloblastoma. Ependymoma prediction was the least accurate. Tumor type classification accuracy and F 1 score were higher than those of 2 of the 4 radiologists., Conclusions: We present a multi-institutional deep learning model for pediatric posterior fossa tumor detection and classification with the potential to augment and improve the accuracy of radiologic diagnosis., (© 2020 by American Journal of Neuroradiology.)

Introduction: Beyond focal radiation, there is no consensus standard therapy for pediatric high-grade glioma (pHGG) and outcomes remain dismal. We describe the largest molecularly-characterized cohort of children with pHGG treated with a 3-drug maintenance regimen of temozolomide, irinotecan, and bevacizumab (TIB) following radiation., Methods: We retrospectively reviewed 36 pediatric patients treated with TIB at Seattle Children's Hospital from 2009 to 2018 and analyzed survival using the Kaplan-Meier method. Molecular profiling was performed by targeted DNA sequencing and toxicities, steroid use, and palliative care utilization were evaluated., Results: Median age at diagnosis was 10.9 years (18 months-18 years). Genetic alterations were detected in 26 genes and aligned with recognized molecular subgroups including H3 K27M-mutant (12), H3F3A G34-mutant (2), IDH-mutant (4), and hypermutator profiles (4). Fifteen patients (42%) completed 12 planned cycles of maintenance. Side effects associated with chemotherapy delays or modifications included thrombocytopenia (28%) and nausea/vomiting (19%), with temozolomide dosing most frequently modified. Median event-free survival (EFS) and overall survival (OS) was 16.2 and 20.1 months, with shorter survival seen in DIPG (9.3 and 13.3 months, respectively). Survival at 1, 2, and 5 years was 80%, 10% and 0% for DIPG and 85%, 38%, and 16% for other pHGG., Conclusion: Our single-center experience demonstrates tolerability of this 3-drug regimen, with prolonged survival in DIPG compared to historical single-agent temozolomide. pHGG survival was comparable to analogous 3-drug regimens and superior to historical agents; however, cure was rare. Children with pHGG remain excellent candidates for the study of novel therapeutics combined with standard therapy.

Purpose: Intracranial growing teratoma syndrome (iGTS) is a rare phenomenon of paradoxical growth of a germ cell tumor (GCT) during treatment despite normalization of tumor markers. We sought to evaluate the frequency, clinical characteristics and outcome of iGTS in Western countries., Methods: Pediatric patients from 22 North American and Australian institutions diagnosed with iGTS between 2000 and 2017 were retrospectively evaluated., Results: From a total of 777 cases of central nervous system (CNS) GCT, 39 cases of iGTS were identified for an overall frequency of 5%. Pineal region was a more frequent location for iGTS as compared to cases of GCT without iGTS (p < 0.00001). In patients with an initial tissue diagnosis of GCT, immature teratoma was present in 50%. Serum AFP or ßhCG was detectable in 87% of patients (median values 66 ng/mL and 44 IU/L, respectively). iGTS occurred at a median of 2 months (range 0.5-32) from diagnosis, in the majority of patients. All patients underwent surgical resection, leading to gross total resection in 79%. Following surgery, all patients resumed adjuvant therapy or post treatment follow-up for GCT. At a median follow-up of 5.3 years (range 0.2-11.8), 37 (95%) of patients are alive, including 5 with stable residual mass., Conclusion: iGTS occurs in 5% of patients with GCT in Western countries. Tumors of the pineal region and GCT containing immature teratoma appear to be associated with a higher risk of developing iGTS. Complete surgical resection is the mainstay of treatment. Overall survival of patients developing iGTS remains favorable.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brainstem tumor affecting approximately 300 children in the US annually. Median survival is less than 1 year, and radiation therapy has been the mainstay of treatment for decades. Recent advances in the biological understanding of the disease have identified the H3K27M mutation in nearly 80% of DIPGs, leading to the 2016 WHO classification of diffuse midline glioma H3K27M-mutant, a grade IV brainstem tumor. Developments in epigenetic targeting of transcriptional tendencies have yielded potential molecular targets for clinical trials. Chimeric antigen receptor T cell therapy has also shown preclinical promise. Recent clinical studies, including prospective trials, have demonstrated the safety and feasibility of pediatric brainstem biopsy in the setting of DIPG and other brainstem tumors. Given developments in the ability to analyze DIPG tumor tissue to deepen biological understanding of this disease and develop new therapies for treatment, together with the increased safety of stereotactic brainstem biopsy, the authors present a case for offering biopsy to all children with suspected DIPG. They also present their standard operative techniques for image-guided, frameless stereotactic biopsy.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells., Competing Interests: CONFLICTS OF INTEREST Dr Orentas conducts consulting and sponsored research for Miltenyi Biotec, Inc. Dr Jensen has received grants and personal fees from Juno Therapeutics, a Celgene Company, during the conduct of the study but outside the submitted work. In addition, Dr Jensen holds a patent “Defined Composition Gene Modified T Cell Products”, with royalties paid to Juno Therapeutics. The remaining authors declare no conflicts of interest., (Copyright: © 2019 Ravanpay et al.)

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD + ) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Niklas KrummStock and Other Ownership Interests: Reference GenomicsAndrew W. StaceyHonoraria: Immunocore Travel, Accommodations, Expenses: Immunocore No other potential conflicts of interest were reported.

Purpose of Review: This review of diffuse intrinsic pontine glioma (DIPG) provides clinical background, a systematic approach to diagnosis and initial care, and synthesizes historical, modern, and future directions for treatment. We present evidence supporting neurosurgical biopsy, early palliative care involvement, limitation of glucocorticoid use, and the leveraging of preclinical DIPG models as a pipeline to next-generation clinical trials., Recent Findings: New molecular understanding of pediatric high-grade gliomas has led to the reclassification of DIPG as one member of a family of diffuse gliomas occurring in the midline of the central nervous system that exhibit pathognomonic mutations in genes encoding histone 3 (H3 K27M). DIPG remains a clinically relevant term, though diagnostically the 80% of DIPG cases that exhibit the H3 K27M mutation have been reclassified as diffuse midline glioma, H3 K27M-mutant. Re-irradiation has been shown to be well-tolerated and of potential benefit. Epigenetic targeting of transcriptional dependencies in preclinical models is fueling molecularly targeted clinical trials. Chimeric antigen receptor T cell immunotherapy has also demonstrated efficacy in preclinical models and provides a promising new clinical strategy. DIPG is a universally fatal, epigenetically driven tumor of the pons that is considered part of a broader class of diffuse midline gliomas sharing H3 K27M mutations. Radiation remains the standard of care, single-agent temozolomide is not recommended, and glucocorticoids should be used only sparingly. A rapid evolution of understanding in the chromatin, signaling, and immunological biology of DIPG may soon result in clinical breakthroughs.

Background and Purpose: Distinct molecular subgroups of pediatric medulloblastoma confer important differences in prognosis and therapy. Currently, tissue sampling is the only method to obtain information for classification. Our goal was to develop and validate radiomic and machine learning approaches for predicting molecular subgroups of pediatric medulloblastoma., Materials and Methods: In this multi-institutional retrospective study, we evaluated MR imaging datasets of 109 pediatric patients with medulloblastoma from 3 children's hospitals from January 2001 to January 2014. A computational framework was developed to extract MR imaging-based radiomic features from tumor segmentations, and we tested 2 predictive models: a double 10-fold cross-validation using a combined dataset consisting of all 3 patient cohorts and a 3-dataset cross-validation, in which training was performed on 2 cohorts and testing was performed on the third independent cohort. We used the Wilcoxon rank sum test for feature selection with assessment of area under the receiver operating characteristic curve to evaluate model performance., Results: Of 590 MR imaging-derived radiomic features, including intensity-based histograms, tumor edge-sharpness, Gabor features, and local area integral invariant features, extracted from imaging-derived tumor segmentations, tumor edge-sharpness was most useful for predicting sonic hedgehog and group 4 tumors. Receiver operating characteristic analysis revealed superior performance of the double 10-fold cross-validation model for predicting sonic hedgehog, group 3, and group 4 tumors when using combined T1- and T2-weighted images (area under the curve = 0.79, 0.70, and 0.83, respectively). With the independent 3-dataset cross-validation strategy, select radiomic features were predictive of sonic hedgehog (area under the curve = 0.70-0.73) and group 4 (area under the curve = 0.76-0.80) medulloblastoma., Conclusions: This study provides proof-of-concept results for the application of radiomic and machine learning approaches to a multi-institutional dataset for the prediction of medulloblastoma subgroups., (© 2019 by American Journal of Neuroradiology.)