Comparison of Volumetric and 2D Measurements and Longitudinal Trajectories in the Response Assessment of BRAF V600E-Mutant Pediatric Gliomas in the Pacific Pediatric Neuro-Oncology Consortium Clinical Trial.

BACKGROUND AND PURPOSE: Response on imaging is widely used to evaluate treatment efficacy in clinical trials of pediatric gliomas. While conventional criteria rely on 2D measurements, volumetric analysis may provide a more comprehensive response assessment. There is sparse research on the role of volumetrics in pediatric gliomas. Our purpose was to compare 2D and volumetric analysis with the assessment of neuroradiologists using the Brain Tumor Reporting and Data System (BT-RADS) in BRAF V600E-mutant pediatric gliomas. MATERIALS AND METHODS: Manual volumetric segmentations of whole and solid tumors were compared with 2D measurements in 31 participants (292 follow-up studies) in the Pacific Pediatric Neuro-Oncology Consortium 002 trial (NCT01748149). Two neuroradiologists evaluated responses using BT-RADS. Receiver operating characteristic analysis compared classification performance of 2D and volumetrics for partial response. Agreement between volumetric and 2D mathematically modeled longitudinal trajectories for 25 participants was determined using the model-estimated time to best response. RESULTS: Of 31 participants, 20 had partial responses according to BT-RADS criteria. Receiver operating characteristic curves for the classification of partial responders at the time of first detection (median = 2 months) yielded an area under the curve of 0.84 (95% CI, 0.69-0.99) for 2D area, 0.91 (95% CI, 0.80-1.00) for whole-volume, and 0.92 (95% CI, 0.82-1.00) for solid volume change. There was no significant difference in the area under the curve between 2D and solid (P = .34) or whole volume (P = .39). There was no significant correlation in model-estimated time to best response (ρ = 0.39, P >.05) between 2D and whole-volume trajectories. Eight of the 25 participants had a difference of ≥90 days in transition from partial response to stable disease between their 2D and whole-volume modeled trajectories. CONCLUSIONS: Although there was no overall difference between volumetrics and 2D in classifying partial response assessment using BT-RADS, further prospective studies will be critical to elucidate how the observed differences in tumor 2D and volumetric trajectories affect clinical decision-making and outcomes in some individuals.

Targeted gene expression profiling predicts meningioma outcomes and radiotherapy responses.

Surgery is the mainstay of treatment for meningioma, the most common primary intracranial tumor, but improvements in meningioma risk stratification are needed and indications for postoperative radiotherapy are controversial. Here we develop a targeted gene expression biomarker that predicts meningioma outcomes and radiotherapy responses. Using a discovery cohort of 173 meningiomas, we developed a 34-gene expression risk score and performed clinical and analytical validation of this biomarker on independent meningiomas from 12 institutions across 3 continents (N = 1,856), including 103 meningiomas from a prospective clinical trial. The gene expression biomarker improved discrimination of outcomes compared with all other systems tested (N = 9) in the clinical validation cohort for local recurrence (5-year area under the curve (AUC) 0.81) and overall survival (5-year AUC 0.80). The increase in AUC compared with the standard of care, World Health Organization 2021 grade, was 0.11 for local recurrence (95% confidence interval 0.07 to 0.17, P < 0.001). The gene expression biomarker identified meningiomas benefiting from postoperative radiotherapy (hazard ratio 0.54, 95% confidence interval 0.37 to 0.78, P = 0.0001) and suggested postoperative management could be refined for 29.8% of patients. In sum, our results identify a targeted gene expression biomarker that improves discrimination of meningioma outcomes, including prediction of postoperative radiotherapy responses.

Advanced Age in Humans and Mouse Models of Glioblastoma Show Decreased Survival from Extratumoral Influence.

PURPOSE: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. EXPERIMENTAL DESIGN: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. RESULTS: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. CONCLUSIONS: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

Targeted gene expression profiling predicts meningioma outcomes and radiotherapy responses.

Background: Surgery is the mainstay of treatment for meningioma, the most common primary intracranial tumor, but improvements in meningioma risk stratification are needed and current indications for postoperative radiotherapy are controversial. Recent studies have proposed prognostic meningioma classification systems using DNA methylation profiling, copy number variants, DNA sequencing, RNA sequencing, histology, or integrated models based on multiple combined features. Targeted gene expression profiling has generated robust biomarkers integrating multiple molecular features for other cancers, but is understudied for meningiomas. Methods: Targeted gene expression profiling was performed on 173 meningiomas and an optimized gene expression biomarker (34 genes) and risk score (0 to 1) was developed to predict clinical outcomes. Clinical and analytical validation was performed on independent meningiomas from 12 institutions across 3 continents (N = 1856), including 103 meningiomas from a prospective clinical trial. Gene expression biomarker performance was compared to 9 other classification systems. Results: The gene expression biomarker improved discrimination of postoperative meningioma outcomes compared to all other classification systems tested in the independent clinical validation cohort for local recurrence (5-year area under the curve [AUC] 0.81) and overall survival (5-year AUC 0.80). The increase in area under the curve compared to the current standard of care, World Health Organization 2021 grade, was 0.11 for local recurrence (95% confidence interval [CI] 0.07-0.17, P < 0.001). The gene expression biomarker identified meningiomas benefiting from postoperative radiotherapy (hazard ratio 0.54, 95% CI 0.37-0.78, P = 0.0001) and re-classified up to 52.0% meningiomas compared to conventional clinical criteria, suggesting postoperative management could be refined for 29.8% of patients. Conclusions: A targeted gene expression biomarker improves discrimination of meningioma outcomes compared to recent classification systems and predicts postoperative radiotherapy responses.

Reversal of cancer gene expression identifies repurposed drugs for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.

A Holistic Review on the Current and Future Status of Biology-Driven and Broad-Spectrum Therapeutic Options for Medulloblastoma.

With a thorough investigation of the etiology of medulloblastomas, a comprehensive review was done to categorize available clinical trials in order to discuss the future potential of breakthroughs in treatment options. The pertinent issues of medulloblastoma therapy with radiation being inapplicable to children under the age of 3, and therapies causing toxicity are detailed and discussed in the context of understanding how the current therapies may address these suboptimal treatment modalities. This study aggregated published studies from the US government clinical trials website and filtered them based on their direct treatment towards medulloblastomas. Thirty-two clinical trials were applicable to be analyzed and the treatment mechanisms were discussed along with the efficacy; molecular groupings of medulloblastomas were also investigated. The investigated therapies tend to target sonic hedgehog (SHH)-subtype medulloblastomas, but there is a necessity for group 3 subtype and group 4 subtype to be targeted as well. Due to the heterogeneous nature of tumor relapse in groups 3 and 4, there are less specified trials towards those molecular groupings, and radiation seems to be the main scope of treatment. Medulloblastomas being primarily a pediatric tumor require treatment options that minimize radiation to increase the quality of living in children and to prevent long-term symptoms of over radiation. Exploring symptomatic treatment with donepezil in children with combination therapies may be a potential route for future trials; immunotherapies seem to hold potential in treating patients reacting adversely to radiation therapy.

Tumor-Educated Platelets: A Review of Current and Potential Applications in Solid Tumors.

In this current era of precision medicine, liquid biopsy poses a unique opportunity for an easily accessible, comprehensive molecular profile that would allow for the identification of therapeutic targets and sequential monitoring. Solid tumors are definitively diagnosed by analyzing primary tumor tissue, but surgical sampling is not always sufficient to generate a comprehensive genetic fingerprint at the time of diagnosis, or an appropriate means for continued monitoring. Platelets are known to have a dynamic, bidirectional relationship with tumors, acting beyond their role of hemostasis. Tumor-educated platelets (TEP) are modified by the tumor in multiple ways and act as a carrier and protector of metastasis. Data so far have shown that the mRNA in TEP can be harnessed for cancer diagnostics, with many potential applications.

A Novel Blood-Brain Barrier-Permeable Chemotherapeutic Agent for the Treatment of Glioblastoma.

Introduction The standard treatment for glioblastoma (GBM) patients is surgical tumor resection, followed by radiation and chemotherapy with temozolomide (TMZ). Unfortunately, 60% of newly diagnosed GBM patients express high levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) and are TMZ-resistant, and all patients eventually become refractory to treatment. The blood-brain barrier (BBB) is an obstacle to the delivery of chemotherapeutic agents to GBM, and BBB-permeable agents that are efficacious in TMZ-resistant and refractory patients are needed. The large amino acid transporter 1 (LAT1) is expressed on the BBB and in GBM and is detected at much lower levels in normal brain tissue. A LAT1-selective therapeutic would potentially target brain tumors while avoiding uptake by healthy tissue. Methods We report a novel chemical entity (QBS10072S) that combines a potent cytotoxic chemotherapeutic domain (tertiary N-bis(2-chloroethyl)amine) with the structural features of a selective LAT1 substrate and tested it against GBM models in vitro and in vivo. For in vitro studies, DNA damage was assessed with a gamma H2A.X antibody and cell viability was assessed by WST-1 assay and/or CellTiter-Glo assay. For in vivo studies, QBS10072S (with or without radiation) was tested in orthotopic glioblastoma xenograft models, using overall survival and tumor size (as measured by bioluminescence), as endpoints. Results QBS10072S is 50-fold more selective for LAT1 vs. LAT2 in transport assays and demonstrates significant growth suppression in vitro of LAT1-expressing GBM cell lines. Unlike TMZ, QBS10072S is cytotoxic to cells with both high and low levels of MGMT expression. In orthotopic GBM xenografts, QBS10072S treatment significantly delayed tumorigenesis and prolonged animal survival compared to the vehicle without adverse effects. Conclusion QBS10072S is a novel BBB-permeable chemotherapeutic agent with the potential to treat TMZ-resistant and recurrent GBM as monotherapy or in combination with radiation treatment.

Deconvoluting Mechanisms of Acquired Resistance to RAF Inhibitors in BRAFV600E-Mutant Human Glioma.

PURPOSE: Selective RAF-targeted therapy is effective in some patients with BRAFV600E-mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms. EXPERIMENTAL DESIGN: Paired pre-/post- RAF inhibitor (RAFi)-treated glioma samples (N = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAFV600E-mutant glioma cell lines. RESULTS: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1, and MAP2K1). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN, or CBL, in BRAFV600E-mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes. CONCLUSIONS: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach.

Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma.

Pediatric and adult high-grade gliomas (HGGs) frequently harbor PDGFRA alterations. We hypothesized that cotreatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib. We performed dose-response, synergism, P-glycoprotein inhibition, and pharmacokinetic studies in in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus. We found that dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with a reduction in mTOR signaling that persisted after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended the survival of PPK tumor-bearing mice (mutant TP53, mutant PDGFRA, H3K27M). Six pediatric patients with glioma tolerated this combination without significant adverse events, and 4 patients with recurrent disease (n = 4) had a median overall survival of 8.5 months. Our results show that the efficacy of dasatinib treatment of PDGFRα-driven HGG was enhanced with everolimus and suggest a promising route for improving targeted therapy for this patient population.

Phase I study of vemurafenib in children with recurrent or progressive BRAFV600E mutant brain tumors: Pacific Pediatric Neuro-Oncology Consortium study (PNOC-002).

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).

Population Pharmacokinetics of Vemurafenib in Children With Recurrent/Refractory BRAF Gene V600E-Mutant Astrocytomas.

Vemurafenib (Zelboraf) is an orally available BRAFV600E inhibitor approved for the treatment of unresectable or metastatic BRAFV600E -mutant melanoma. The primary objective of this work was to characterize the pharmacokinetics (PK) of vemurafenib in pediatric patients with recurrent/refractory astrocytomas harboring the BRAFV600E mutation. The study was also designed to evaluate the feasibility of replacing whole vemurafenib tablets with crushed tablets in young children unable to swallow tablets. Twenty-five pediatric patients (median age, 8.8 years; range, 3.3-19.2) with recurrent/refractory BRAFV600E -mutant astrocytomas received whole (n = 19) or crushed (n = 6) vemurafenib tablets twice daily. Plasma samples were collected on days 1, 15, and 22 in cycle 1 of vemurafenib treatment. Descriptive PK analyses demonstrated significant variability (approximately 6-fold) in drug exposure. A 1-compartment model with first-order absorption and elimination was developed by adjusting the vemurafenib PK model previously validated in adults with mutant BRAFV600E melanoma. After inclusion of allometric scaling on total body weight, the model adequately described the PK of vemurafenib in children between a wide age range of 3 to 19 years old. In the crushed-tablet cohort, relative bioavailability was approximately 96% (95% confidence interval, 49%-142%) compared to that seen in pediatric patients receiving whole tablets based on the preliminary comparison analysis results. Moderate intrapatient variability (48%) of vemurafenib clearance was observed. There was significant correlation (R2 = 0.83) between area under the plasma concentration-time curve and trough concentration at steady state. These results will help increase the number of pediatric patients for whom vemurafenib is accessible and facilitate improved dosing in pediatric patients with recurrent/refractory BRAFV600E astrocytomas.

Exploring DNA Methylation for Prognosis and Analyzing the Tumor Microenvironment in Pleomorphic Xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a rare type of brain tumor that affects children and young adults. Molecular prognostic markers of PXAs remain poorly established. Similar to gangliogliomas, PXAs show prominent immune cell infiltrate, but its composition also remains unknown. In this study, we correlated DNA methylation and BRAF status with clinical outcome and explored the tumor microenvironment. We performed DNA methylation in 21 tumor samples from 18 subjects with a histological diagnosis of PXA. MethylCIBERSORT was used to deconvolute the PXA microenvironment by analyzing the associated immune cell-types. Median age at diagnosis was 16 years (range 7-32). At median follow-up of 30 months, 3-year and 5-year overall survival was 73% and 71%, respectively. Overall survival ranged from 1 to 139 months. Eleven out of 18 subjects (61%) showed disease progression. Progression-free survival ranged from 1 to 89 months. Trisomy 7 and CDKN2A/B (p16) homozygous deletion did not show any association with overall survival (p = 0.67 and p = 0.74, respectively). Decreased overall survival was observed for subjects with tumors lacking the BRAF V600E mutation (p = 0.02). PXAs had significantly increased CD8 T-cell epigenetic signatures compared with previously profiled gangliogliomas (p = 0.0019). The characterization of immune cell-types in PXAs may have implications for future development of immunotherapy.

Germline MUTYH Mutation in a Pediatric Cancer Survivor Developing a Secondary Malignancy.

Radiotherapy-induced second malignant neoplasms (SMNs) are a severe late complication in pediatric cancer survivors. Germline mutations in tumor suppressor genes contribute to SMNs; however, the most relevant germline variants mediating susceptibility are not fully defined. The authors performed matched whole-exome sequencing analyses of germline and tumor DNA from 4 pediatric solid tumor survivors who subsequently developed radiation-associated SMNs. Pathogenic and predicted deleterious germline variants were identified for each patient and validated with Sanger sequencing. These germline variants were compared with germline variants in a cohort of 59 pediatric patients diagnosed with primary sarcomas. Pathway analysis was performed to test for similarities in the germline variant profiles between individuals diagnosed with SMNs or primary sarcomas. One index patient was found to have a pathogenic germline monoallelic mutation in the MUTYH gene, which encodes the base excision repair enzyme adenine DNA glycosylase. This specific germline mutation is associated with a form of familial adenomatous polyposis, a new diagnosis in the patient. Germline-level genetic similarity exists between SMN-developing patients and patients developing primary sarcomas, with relevant genes involved in signal transduction and DNA repair mechanisms. The authors identify a germline MUTYH mutation in a pediatric cancer survivor developing an SMN. Germline mutations involving specific pathways such as base excision repair may identify individuals at risk for developing SMNs. The composition of germline variants in individual patients may enable estimates of patient-specific risk for developing SMNs. The authors anticipate that further analyses of germline genomes and epigenomes will reveal diverse genes and mechanisms influencing cancer risk.

Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

Mechanisms of Resistance to EGFR Inhibition Reveal Metabolic Vulnerabilities in Human GBM.

Amplification of the epidermal growth factor receptor gene (EGFR) represents one of the most commonly observed genetic lesions in glioblastoma (GBM); however, therapies targeting this signaling pathway have failed clinically. Here, using human tumors, primary patient-derived xenografts (PDX), and a murine model for GBM, we demonstrate that EGFR inhibition leads to increased invasion of tumor cells. Further, EGFR inhibitor-treated GBM demonstrates altered oxidative stress, with increased lipid peroxidation, and generation of toxic lipid peroxidation products. A tumor cell subpopulation with elevated aldehyde dehydrogenase (ALDH) levels was determined to comprise a significant proportion of the invasive cells observed in EGFR inhibitor-treated GBM. Our analysis of the ALDH1A1 protein in newly diagnosed GBM revealed detectable ALDH1A1 expression in 69% (35/51) of the cases, but in relatively low percentages of tumor cells. Analysis of paired human GBM before and after EGFR inhibitor therapy showed an increase in ALDH1A1 expression in EGFR-amplified tumors (P < 0.05, n = 13 tumor pairs), and in murine GBM ALDH1A1-high clones were more resistant to EGFR inhibition than ALDH1A1-low clones. Our data identify ALDH levels as a biomarker of GBM cells with high invasive potential, altered oxidative stress, and resistance to EGFR inhibition, and reveal a therapeutic target whose inhibition should limit GBM invasion.

Molecular profiling and targeted therapy in pediatric gliomas: review and consensus recommendations.

As the field of neuro-oncology makes headway in uncovering the key oncogenic drivers in pediatric glioma, the role of precision diagnostics and therapies continues to rapidly evolve with important implications for the standard of care for clinical management of these patients. Four studies at major academic centers were published in the last year outlining the clinically integrated molecular profiling and targeting of pediatric brain tumors; all 4 demonstrated the feasibility and utility of incorporating sequencing into the care of children with brain tumors, in particular for children and young adults with glioma. Based on synthesis of the data from these studies and others, we provide consensus recommendations for the integration of precision diagnostics and therapeutics into the practice of pediatric neuro-oncology. Our primary consensus recommendation is that next-generation sequencing should be routinely included in the workup of most pediatric gliomas.