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

Clinical information for PNOC003 plasma ctDNA studies.

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

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

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

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

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

Molecular characteristics of pediatric brain tumors have not only allowed for tumor subgrouping but have introduced novel treatment options for patients with specific tumor alterations. Therefore, an accurate histologic and molecular diagnosis is critical for optimized management of all pediatric patients with brain tumors, including central nervous system embryonal tumors. We present a case where optical genome mapping identified a ZNF532-NUTM1 fusion in a patient with a unique tumor best characterized histologically as a central nervous system embryonal tumor with rhabdoid features. Additional analyses including immunohistochemistry for NUT protein, methylation array, whole genome, and RNA-sequencing was done to confirm the presence of the fusion in the tumor. This is the first description of a pediatric patient with a ZNF532-NUTM1 fusion, yet the histology of this tumor is similar to that of adult cancers with ZNF-NUTM1 fusions and other NUTM1-fusion positive brain tumors reported in literature. Although rare, the distinct pathology and underlying molecular characteristics of these tumors separate them from other embryonal tumors. Therefore, the NUTM-rearrangement appears to define a novel subgroup of pediatric central nervous system embryonal tumors with rhabdoid/epithelioid features that may have a unique response to treatment. Screening for a NUTM1-rearrangement should be considered for all patients with unclassified central nervous system tumors with rhabdoid features to ensure accurate diagnosis so this can ultimately inform therapeutic management for these patients.

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

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

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

INTRODUCTION: Diffuse midline glioma (DMG) is a fatal childhood CNS tumor. Magnetic resonance imaging (MRI) is the gold standard for DMG diagnosis and monitoring of response to therapy. Leveraging novel MRI analytical approaches, including volumetric and machine learning based analyses, may aid in the prediction of patient overall survival (OS) and help to identify high-risk cases. METHODS: T1- and T2-weighted MR images were retrospectively collected from children and young adults diagnosed with DMG (n=43). MRI features, including manually determined 3D tumor volume (T2), T1 contrast-enhancing tumor volume, T1 relative to T2 volume (T1/T2), tumor relative to whole brain volume, tumor average intensity, and tumor heterogeneity (i.e., intensity skewness and kurtosis), were evaluated at upfront diagnosis. MRI features were analyzed to identify significant predictors of OS outcome, which was defined as OS shorter, or longer, than one year from diagnosis. A support vector machine was used to predict OS outcomes using combinations of these features. RESULTS: The presence of T1 contrast-enhancing tumor at diagnosis (p=0.01), and a high T1/T2 ratio (>25%, p=0.009), predicted significantly shorter median OS. Moreover, feature selection identified T2 mean intensity (p

Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

Background Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. Methods ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. Results ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. Conclusion Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly tumors comprising 10%–15% of all childhood CNS cancers. Standard treatment is considered palliative and prognosis is near universal mortality. DIPGs have been classified into genomic subtypes based on histone variants with the lysine to methionine mutation on position 27 of histone tails (K27M). Given the increasing promise of immunotherapy, there have been ongoing efforts to identify tumor-specific antigens to serve as immunologic targets. We evaluated a large cohort of CNS specimens for Wilms’ tumor protein (WT1) expression. These specimens include primary pediatric CNS tumors (n = 38 midline gliomas and n = 3 non-midline gliomas; n = 23 DIPG, n = 10 low-grade gliomas, n = 8 high-grade gliomas), and DIPG primary cells. Here, we report the validation of WT1 as a tumor-associated antigen in DIPGs. We further report that WT1 expression is significantly correlated with specific oncohistone variants, with the highest expression detected in the H3.3K27M subgroup. WT1 expression was absent in all control specimens (n = 21). Western blot assays using DIPG primary cells (n = 6) showed a trend of higher WT1 expression in H3.3K27M cells when compared with H3.1 K27M cells and H3 wildtype cells. Our data are the first indication of the association between WT1 and DIPG, with specific upregulation in those harboring oncohistone H3.3K27M.

RATIONALE: One of the major hurdles in developing effective treatment for children with DIPG includes the lack of extensive combinatorial studies targeting major driver oncogenic pathways. Combination of H3K27M and TP53 mutations are found in over 50% of DIPG tumors, however, there is a lack of effective combinatorial precision therapy. METHODS: Tissue specimens obtained from subjects with DIPG were used to generate preclinical models. Subjects were enrolled in a prospective molecular diagnostic clinical trial and specimens were obtained by surgical biopsy both at initial diagnosis, and at the time of progression. RESULTS: Fourteen primary neurosphere cell lines and nine xenograft mouse models were successfully generated. The success rate of generating primary neurospheres and xenograft models was 45% and 75%, respectively. Among the 14 cell lines, one was derived from a biopsy performed after tumor progression, and one from washing a biopsy needle. Whole genome analysis of three biopsy-derived primary neurospheres revealed that these samples shared genomic alterations when compared to the primary tumors. All cell lines retained major oncogenic driver mutations in genes such as H3F3A, HIST1H3B, TP53, PPM1D, PIK3R1, and ACVR1. The needle-wash derived cell line was immortalized with hTERT, and exhibited the most deviation in mutation profiles and copy number alterations compared to primary tumor. Global DNA methylation profiling of seven primary tumor biopsies and matched primary neurospheres revealed similar epigenetic profiles. Primary neurospheres derived from biopsies were treated with the same specialized panel of agents recommended by a personalized medicine tumor board, which was based on the mutational profiles of the original tumor. SIGNIFICANCE: All cell lines exhibited varying levels of sensitivity to single agent indicating patterns of intertumor heterogeneity. The use of these model systems allows testing of combinatorial precision therapy for patients with DIPG, where there is a need for rapid translation into clinical trials.

INTRODUCTION: Molecular profiling of the tumor genome is critical for diagnosis and clinical management of CNS tumors. In the case of pediatric diffuse midline gliomas (DMGs), the neuroanatomical location of the tumor precludes surgical resection. Moreover, when tumor resection or biopsy is permitted, serial monitoring of the tumor is limited to MR imaging. We have recently reported on detection of tumor associated somatic mutations in patient liquid biopsy specimens. While powerful, our current approach is limited to multiplexed detection of up to two somatic mutations, limiting comprehensive profiling. To address this, we developed a sensitive approach for profiling tumor genomic aberrations in circulating tumor DNA (ctDNA). RESULTS: We show successful detection of tumor-specific mutations in plasma, serum, and cerebrospinal fluid (CSF) of pediatric DMGs, using a targeted panel to sequence exons of 170 candidate genes. Our analysis indicates overlap of somatic tumor mutations in CSF and plasma with matched tissue; a higher number of mutations were detected in CSF compared to plasma. Patterns of tumor evolution were revealed in CSF analyzed at end-stage disease. For example, loss of a TP53 mutation at progression compared to initial diagnosis was confirmed in CSF collected at postmortem, indicating utility of our platform for identification of tumor evolution. Additionally, subclonal heterogeneity of DMGs was captured in CSF compared to tumor specimens. Low allelic frequency mutations in genes involved in DMG tumorigenesis, such as PI3K signaling and chromatin regulators, were exclusively detected in CSF, but not in matched tumor tissue. IMPACT: Our novel approach enables tumor exome monitoring of low frequency variants in liquid biopsy specimens. Our sensitive targeted sequencing panel provides opportunity for profiling somatic tumor mutations at initial diagnosis and at progression, for mapping tumor evolution in the context of clinical trials.

In order to define the mutational landscape across histone genes in pediatric CNS cancers, we surveyed 1,000 patients from the Children’s Brain Tumor Tissue Consortium (CBTTC) cohort, representing 1,100 samples from 35 studies. In addition, we amassed genomic data from 152 pediatric high grade glioma subjects (114 WGS, 38 WES) representing both upfront biopsies and autopsy specimens. Using existing and newly generated genomic data, we comprehensively mapped the histone mutation landscape in pediatric gliomas. We identified previously unknown and novel histone mutations, mostly associated with midline gliomas (pontine and thalamic). Mutations were classified as protein damaging (causing a change in amino acid or frameshift), rare (present in less than 0.01% of the population), and somatic by comparison to matched germline DNA, or to a large panel of normal brain samples from the CBTTC cohort (n=40). Mutations were found in both linker and core histone genes, validated by Sanger sequencing, and defined in the context of their clinical impact. The majority of these mutations are expected to decrease protein stability, and to perturb post-translational modifications (in particular methylation and/or phosphorylation) at the protein level. Novel mutations were identified in tumor samples harboring K27M in H3.3 and H3.1, as well as in tumors classified as histone wild type (due to absence of K27M mutation). Subjects harboring novel histone mutations were found to have higher overall mutational load, and unsupervised clustering of methylation data has identified distinct epigenetic patterns within H3.3/3.1 K27M subjects with and without novel histone mutations. We also assessed co-occurrence of novel histone mutations with known partner mutations, and identified genes encoding DNA-binding proteins significantly associated with novel linker histone mutations. In vitro studies are underway to define the biological role of novel histone mutations in tumorigenicity.

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

Diffuse intrinsic pontine glioma (DIPG) is a deadly pediatric brain cancer constituting 10–15% of all central nervous system (CNS) tumors in children. Its anatomical location and infiltrative nature make it one of the most challenging tumors to treat. As such, targeted therapies are gaining more interest in CNS tumors. To identify a suitable therapeutic target, we evaluated publicly available gene expression data of pediatric glioblastoma specimens (n=34, GSE50161), DIPG specimens (n=35, GSE50021) and gene expression data of DIPG specimens from our previous dataset (n=5). Further analysis using 37 pediatric midline glioma specimens collected at autopsy in accordance with Children’s National Health System Institutional Review Board approval, indicated overexpression of one candidate protein, Wilms’ tumor protein (WT1), by immunohistochemistry. Interestingly, our immunohistochemistry and immunofluorescence analysis showed that WT1 in DIPG specimens is localized in the cytoplasm. Real-Time Quantitative Reverse Transcription PCR analysis using fresh frozen tumor specimens also showed that H3.3K27M-DIPG subtypes (n=3) to express higher levels of WT1 protein compared to those with H3.1K27M (n=2) (5.3 fold). More importantly, RNA-seq analysis of DIPG tumors (n=23) revealed specific transcriptome expression of WT1 transcript variant F. Nuclear export signal prediction analysis of the WT1 protein sequence translated by this transcript showed a higher nuclear export signal prediction score compared to other isoforms of WT1. Our findings suggest that WT1, particularly WT1 variant F, is a robust tumor specific antigen in DIPG, which may reveal its biological role in tumorigenesis and may have implications for clinical translation as an immunotherapeutic target.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) has one of the lowest overall survival amongst pediatric brain cancers. DIPG can be stratified into molecular subtypes dictated by histone mutational status: H3.3K27M, H3.1K27M, and H3 wildtype. Mutant histone proteins drive cellular proliferation by reprogramming epigenetic landscape. To interrogate the DIPG epigenome, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in 5 DIPG tumors (H3.3K27M and H3 wildtype) and 3 healthy pons. This is the first report of ChIP-seq in DIPG tissue with H3 wildtype tumor. METHODS: ChIP-Seq was performed on DIPG tumors and control healthy pons for histone modifications indicative of active promoter (H3K4me3), repressive promoter (H3K27me3), and enhancer chromatin (H3K4me1 and H3K27ac). We focused on targets active in both H3K27M and H3 wildtype. Already existing RNA-seq data from DIPG upfront biopsy specimens (n=25) were used to narrow targets with increased transcripts. Immunohistochemistry (IHC) was performed to show protein expression. Human DIPG primary cell lines were assessed using Western blot analysis. RESULTS: Multiple targets were identified from ChIP-seq with activated histone including Notch1, CSPG4, and B7-H3. Focusing on B7-H3, RNA-seq confirmed high transcript levels at 43 Transcripts Per Million (TPM) in DIPG tumor and was significantly elevated compared to adjacent healthy brain at 5.5 TPM (p < 6x10-6). IHC analysis of 3 DIPGs showed strong B7-H3 staining compared to adjacent normal brain. To show the fidelity of preclinical models to human donor, primary DIPG cell lines (n=6) were interrogated by western blot assay. A 4.9 fold increase in B7-H3 (p=0.011) in DIPG cell lines was found when compared to protein lysates from healthy brain tissue (n=3). CONCLUSION: Using primary DIPG tumor tissue for ChIP-Seq analysis is novel and our study shows that epigenetic analysis by ChIP-seq coupled with transcriptional analysis by RNA-seq can be utilized to find new targets for DIPG therapy.

There is a growing role for mutations affecting histone linker and histone core-encoding genes across several adult and pediatric cancers. However, the extent to which somatic histone mutations may bridge across different cancers as common tumorigenic events – particularly in the context of pediatric CNS tumors – remains unclear. To address this knowledge gap, we set out to define a comprehensive pan-cancer landscape of somatic histone mutations. We first queried the ICGC PCAWG and TCGA Pan-Cancer Atlas representing >12,500 adult and pediatric cancer patients. We found lymphomas to be most enriched for histone mutations (50–75%) and, in particular, for mutations in linker histones (HIST1H1B-E), yet also in specific core histone genes (eg, HIST2H2BE). Moreover, we observed a significant enrichment of histone mutations in adult high-grade vs low-grade gliomas (10% vs 6%, P

// Elizabeth Wells 1, 2, * , Madhuri Kambhampati 1, * , Jesse M. Damsker 3, * , Heather Gordish-Dressman 1 , Sridevi Yadavilli 1 , Oren J. Becher 4 , Jamila Gittens 1 , Mojca Stampar 1 , Roger J. Packer 2 , Javad Nazarian 1, 5 1 Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA 2 Brain Tumor Institute, Center for Neuroscience and Behavioral Medicine, Children’s National Health System, Washington, DC, USA 3 ReveraGen BioPharma, Rockville, MD, USA 4 Duke University Medical Center, Durham, NC, USA 5 Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA * These authors contributed equally to this work Correspondence to: Javad Nazarian, email: JNazarian@ChildrensNational.org Keywords: glucocorticoids, cytokines, anti-inflammatory, pre-clinical testing, pediatric brain tumors Received: November 09, 2016 Accepted: December 13, 2016 Published: December 21, 2016 ABSTRACT Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro , murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.

There is no curative treatment for patients with diffuse intrinsic pontine glioma (DIPG). However, with the recent availability of biopsy and autopsy tissue, new data regarding the biologic behavior of this tumor have emerged, allowing greater molecular characterization and leading to investigations which may result in improved therapeutic options. Treatment strategies must address both primary disease sites as well as any metastatic deposits, which may be variably sensitive to a particular approach. In this case report, we present a patient with DIPG treated with irradiation and serial investigational agents. The clinical, pathological and molecular phenotypes of both the progressive primary tumor as well as concomitant metastatic deposits obtained at autopsy are discussed. While some mRNA differences were demonstrated, all analyzed sites of disease shared similar mutational arrangements, suggesting that targeting the mutations of the primary tumor may be effective for all sites of disease.

Nursing teams play an integral role in the care of patients with brain tumors; however nurses do not often see themselves as essential contributors to translational research. Recent development of nurse-researcher relationships and involvement of the multidisciplinary team have led to successful biobanking strategies. Though there are challenges associated with fostering these relationships, their vital role has significantly enhanced participant recruitment and sample collection at one large urban Children’s Hospital. Researchers at the institution have established a biobank to collect samples from pediatric brain tumor patients at diagnosis, during therapy, and post mortem using conventional methods. However, a collaborative environment between nursing and research teams greatly enhanced the growth of the biobank. We have increased patient recruitment by more than 50% in the past four years and supported different types of specimen collection. Our success entails: 1) development of nurse-researcher relationships, 2) an efficient consent process, 3) streamlined sample collection, and 4) appreciation of the vital role of the nursing team in clinical data collection pertinent to molecular analysis. Additionally, the support of nursing is valuable during post mortem consents and provides emotional support to the family to fulfil their wish to donate. Nurses play a major role in coordination of the post-mortem donation process, and assist in the formation of partnerships within the community to promote this opportunity to families. As biobanking continues to be an important part of bench research, all institutions should recognize and support the vital role that nurses can have in enhancing this endeavor.

The Gift from a Child (GFAC) program was inspired by the dream of one child to donate his brain for research, recognizing the need to study tumor tissue collected at diagnosis, recurrence, and at the time of death. Founded by the Swifty Foundation in 2016, GFAC currently is comprised of five “Centers of Excellence” at institutions with expertise in pediatric neuro-oncology. Partnering with the Children’s Brain Tumor Network, the program’s mandate is twofold: make it possible for families to donate no matter where they live in the United States and make tissue available to scientists globally to empower discovery. In order to overcome barriers that have stifled postmortem collection in the past, GFAC has invested in Tissue Navigators - individuals at each center who coordinate all aspects of donation and communicate with families, medical providers, and laboratory scientists. In 2019 alone, GFAC coordinated 55 autopsy collections from multiple diagnosis. A key metric of the program is also capturing the global sharing and usage of each tissue sample, ensuring that tissue isn’t simply “banked” but is actively being actively used to help unravel tumor biology. To date, tissue has been used for genomic and molecular data generation, preclinical model development including cell lines and PDX models, and for novel drug screening. Together with Children’s Brain Tumor Network, the Gift from a Child program is helping to ensure the most precious gift that a family can make is used to accelerate the path to cures.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh frozen DIPG specimens (n=14), normal brain tissue (n=10), and other pediatric brain tumors (n=17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77% of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild type DIPG samples.

Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.

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

Diffuse intrinsic pontine gliomas (DIPG) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DIPG, who have a median survival time of less than one year. Most DIPG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DIPG growth. Suppression of ERK5 decreased DIPG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DIPG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Collectively, our data demonstrate an underappreciated role of H3K27M in RAS activation and reveal novel therapeutic targets for treating DIPG tumors. Significance: These findings identify the H3K27M mutation as an enhancer of RAS activation in DIPG and ERK5 as a novel, immediately actionable molecular target.

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

Diffuse intrinsic pontine glioma (DIPG) is the leading cause of mortality in pediatric central nervous system cancers. Histone mutations are found in >80% of DIPG cases. The most common mutation is histone 3.3 at position 27 resulting in lysine to methionine(H3.3K27M). This mutation results in epigenetic changes in methylation and acetylation of histones leading to aberrant change in transcription globally. Analysis of epigenetic changes via chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been performed in DIPG cell lines but not in tumor tissue. Culture conditions may not accurately reflect the epigenome of the tumor, thus we performed ChIP on three H3.3K27M mutant DIPG tumors (PID675, PID693, PID874). Checking enrichment by PCR revealed that the HOXA gene was enriched by tri-methylation at K27 (K27me3) and TOP3A was enriched by acetylation at K27 (K27ac), as expected. There was enrichment in both K27me3 and K27ac for GLI2. CDKN2A was enriched in K27me3 indicating down regulation in tumor tissue. However, in the DIPG primary cell line derived from PID675, CDKN2A was not enriched in K27me3 or K27ac and GLI2 was only enriched in K27ac. Given the difference seen between tumor tissue and cell line, our findings highlight the crucial importance of profiling the actual tumor tissue in DIPG.

INTRODUCTION: Sharing research ready data between multiple institutions and across different platforms is a lengthy and cumbersome process which can delay research and potentially negatively impact patient care. Despite the recent surge in generating the genomic and molecular profiles of CNS cancers, correlation of this data with clinical or radiographic data is yet to be streamlined. To address this, CBTTC has partnered with Blackfynn, to develop their HIPAA-compliant, cloud-based platform for integration of histopathology, clinical, molecular, and neuroimaging data. This collaboration provides research ready data that can be easily accessed by clinicians and researchers across different institutions. METHODS: MR imaging, genomic, histological data from a cohort of CBTTC patients diagnosed with midline gliomas were used in a pilot trial. The Blackfynn platform was used to share and annotate MR imaging and histological staining across two academic institutions. Clinicians, basic researchers, and software engineers participated in the project with the goal of integrating a multi-disciplinary, cross-data integration platform. RESULTS: The Blackfynn platform successfully allowed sharing and annotation of neuroimages as well as histopathology slides pertaining to the CBTTC cohort. The neuroimaging on the platform is interactive and can be viewed in axial, coronal and sagittal planes. The pathology slides are also interactive and can be viewed up to 50x magnification. Annotations can be made on all of the multimodal data with the possibility of localizing MR images with the corresponding pathology slides. In conclusion, Blackfynn provides the research community research ready data that is easily uploaded and shared between different institutions.

Midline pediatric high grade gliomas (HGGs), especially diffuse intrinsic pontine gliomas (DIPGs), are deadly pediatric brain cancer that makes up 10–15% of all central nervous system (CNS) tumors in children; little progress has been made in improving outcomes for these patients. Immunotherapy is a strategy that has shown promise for treatment of malignancy, although identification of tumor associated antigens is critical to an effective immunotherapeutic approach. Wilms’ tumor protein (WT1) has been heavily implicated in cancer development, and it may serve as an attractive immunotherapy target in HGGs. Here, we validated WT1 as a potential tumor associated antigen in pediatric midline gliomas using formalin fixed paraffin embedded (FFPE) tumor, intra-patient healthy tissue controls, fresh frozen post-mortem tissues, and patient-derived DIPG primary cell lines. Immunohistochemical staining of patient FFPE specimens showed strong WT1 immunoreactivity in tumor compared to adjacent healthy tissue controls. Western blot of tumor further validated higher WT1 levels in tumor versus adjacent normal tissues. Tumor showed cytoplasmic expression of WT1, confirmed by immunofluorescent (IF) staining. In addition, a striking dichotomy of expression was noted with absent/weak WT1 immunoreactivity in H3.1K27M subtype gliomas, compared to moderate/strong signal in H3.3K27M subtypes. Western blotting assay and reverse transcription quantitative PCR using DIPG primary cell lines also validated the differential expression of the protein. Our study suggests that WT1 is an attractive potential target protein for pediatric midline glioma immunotherapy and may differentiate between the underlying histone mutations.

Midline high-grade gliomas (HGGs) are amongst the most challenging childhood cancers to treat. Recent advances in understanding the molecular drivers of these tumors have resulted from donated autopy or biopsy specimens. We have initiated a midline glioma preclinical initiative for producing in vitro and in vivo models that have well defined genomic profiles. We obtained 22 autopsy, 11 upfront biopsy (PNOC NCT 2274987) and 2 surgical resection samples. Collectively, specimens represented DIPGs (n=30) and thalamic HGGs (n=5). Single cell suspensions were generated for neurosphere culture and intracranial injection into mice. Specimens were considered viable if they resulted in two consecutive generations of murine tumor models or five in vitro passages. A total of 14 cell lines representing DIPG and thalamic glioma were generated and validated by short tandem repeat (STR) analysis. Orthotopic injections into non scid gamma (NSG) mice resulted in tumor formation (n=14) with molecular characteristics similar to the corresponding patient tumor. Eleven mouse implantations are still being monitored. In vivo models were validated by immunohistochemical staining for human mitochondria and H3K27M. Methylation and whole genome analysis of preclinical models were generated to assess similarities/shared pathways with human disease. In vitro and in vivo response to therapeutics including panobinostat and TAK228 was also assessed. A microdialysis procedure was optimized to assess blood brain barrier penetration of systemically administered therapeutics. Direct drug delivery was also evaluated using subcutaneously placed osmotic pumps with optimization of delivery rates, cannula depth and placement.

NOTCH regulates stem cells during normal development and stemlike cells in cancer, but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the γ-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, and HES5; inhibited DIPG growth by 75%; and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pretreatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a γ-secretase inhibitor during radiation therapy may be efficacious initially or during reirradiation.

// Sridevi Yadavilli 1 , Joseph Scafidi 2 , Oren J. Becher 3 , Amanda M. Saratsis 4 , Rebecca L. Hiner 5 , Madhuri Kambhampati 1 , Santi Mariarita 6 , Tobey J. MacDonald 7 , Kari-Elise Codispoti 8 , Suresh N. Magge 9 , Jyoti K. Jaiswal 1,10 , Roger J. Packer 11 and Javad Nazarian 1,10 1 Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA 2 Department of Neurology and Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA 3 Department of Pediatrics and Pathology, Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA 4 Division of Neurosurgery, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA 5 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY, USA 6 Department of Pathology and Lab Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA 8 Department of Pathology, Children’s National Health System, Washington, DC, USA 9 Division of Neurosurgery, Children’s National Health System, Washington, DC, USA 10 Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA 11 Brain Tumor Institute, Center for Neuroscience and Behavioral Medicine, Children’s National Health System, Washington, DC, USA Correspondence to: Javad Nazarian, email: // Keywords : DIPG, NG2, PDGF, histone 3, glioma Received : March 04, 2015 Accepted : March 11, 2015 Published : March 30, 2015 Abstract Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50) . We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro . NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

Children diagnosed with diffuse midline histone 3 K27M mutant (H3K27M) glioma continue to have a dismal prognosis with a median survival time of about nine months post-diagnosis. MRI, the standard for assessing tumor growth, is often not sensitive for detecting early tumor regression/progression. We evaluate the utility of biofluids from patients and their xenograft models, for detection of circulating tumor DNA (ctDNA). The goal is to develop a comprehensive panel for ctDNA detection and quantification, capable of assessing response to treatment. Twenty two CSF samples were collected at various stages of disease (upfront, recurrence, and postmortem) from pediatric high grade midline gliomas. Upfront and longitudinal plasma samples (n=68) were collected with each MRI, from 19 diffuse intrinsic pontine glioma (DIPG) patients enrolled in a Phase I clinical trial. For monitoring ctDNA, we developed a liquid biopsy assay using a sensitive and robust digital droplet PCR system on CSF and plasma samples. We successfully detected major DIPG driver mutations including H3F3A, HIST1H3B, and ACVR1. Specifically, we detected H3K27M in 80% of CSF and upfront plasma samples. Our data indicate a concordance of H3K27M in tumor and CSF for 70% of patients (n=13). In accordance with published studies in adult CNS cancers, we detected higher levels of ctDNA in CSF compared to plasma. Longitudinal analysis of plasma showed a drastic decrease in ctDNA following radiotherapy in 86% of patients (n=14), and patients were categorized as responders, where dynamic changes in ctDNA correlated with changes in MRI tumor measurements. Furthermore, we have detected H3K27M in ctDNA of plasma samples in xenograft models of DIPG. We conclude that biofluid is a suitable medium for detection and quantification of ctDNA. Liquid biopsy has the potential to complement, or in some cases eliminate the need for biopsies, and may inform recurrence and tumor response to treatment.

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain cancer which presents an immense therapeutic challenge. Our previous work has shown that histone mutations (H3K27M) arise in the pontine tumor but in most cases necessitates an obligate partner mutation for tumorigenesis and spread. Our established standard operating procedure for performing autopsy has allowed us to collect 20 whole brains from DIPG patients. Sagittal sections from whole brains (n=8) were processed for histological analyses to identify the tumor migratory path (pontine to extra-pontine). Histological examination of all neuroanatomical locations indicated: i) Tumor cell infiltration to extra-pontine regions in 7/8 brains as indicated by histology, ii) In 4/7 brains, tumor cells migrated to extra-pontine regions but failed to form tumors. These regions were histologically “normal” according to the World Health Organization grading criteria, but harbored tumor cells as indicated by H3K27M stains and digital PCR. Given the age of disease onset, and neuroanatomical location, it is plausible that tumor microenvironment plays a critical role in tumor spread. To test the role of pontine microenvironment in tumorigenesis, we performed a pilot study by injecting human pontine tumor cells (n= 4 patients) into 3-4 week old murine cerebral cortex, which represents the age of DIPG disease onset. Necropsy specimens were examined for tumor presence by histological analyses for H&E and H3K27M stains. We identified tumor in both cortex (injection site) and brainstem. Tumor cells migrated to populate brainstem in all four, and cerebellum in 2/4 xenograft models. Furthermore these tumors were positive for human mitochondrial protein (MAB1273) and were authenticated by STR to demonstrate their human cell of origin. These observations strongly show the presence of: i) anatomically suitable migration paths (e.g. axonal guidance, leptomeningeal), and ii) tissue-specific microenvironment and molecular events, governing susceptibility and sensitivity of particular neuroanatomical locations to tumor formation.

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type. Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis. TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.

Pediatric midline gliomas, especially those associated with the pons, known as diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain cancer that makes up 10–15% of all central nervous system (CNS) tumors in children. Its anatomical location and infiltrative nature makes it one of the most challenging tumors to treat. Targeted therapies are gaining more interest in CNS tumors. Identification of tumor associated antigens is one of many requirements in developing an effective targeted therapy. Wilms’ tumor protein (WT1) has been ranked number 1 cancer immunotherapy target by National Cancer Institute. Many types of solid tumors have been shown to express WT1 and it is being examined as one of potential immunotherapeutic targets. Here we validated WT1 as a potential tumor associated antigen in pediatric diffuse midline gliomas using formalin fixed paraffin embedded (FFPE) tumor and adjacent healthy specimens, fresh frozen post-mortem tissues and patient-derived DIPG primary cell lines. Our immunohistochemistry (IHC) staining of patient FFPE specimens showed strong WT1 immunoreactivity in tumor compared to adjacent normal tissue. Western blot of tumor tissues and cell lines were performed to further validate WT1 levels in the tumors versus adjacent healthy tissues. Interestingly, tumors showed cytoplasmic expression of WT1. In addition, H3.1K27M subtype gliomas showed weak to absent WT1 immunoreactivity compared to strong to moderate in H3.3K27M subtypes. Western blots also validated the differential expression of the protein. Our study suggests that WT1 is a potential tumor specific antigen in pediatric midline gliomas, which can be utilized for targeted therapies such as immunotherapy.

INTRODUCTION: Childhood thalamic gliomas are rare cancers, accounting for 5% of all pediatric brain tumors and are of glial origin. The recent WHO classification places thalamic tumors in the category of midline gliomas based on discoveries that indicate histone 3 mutations as the driver genomic aberration of these cancers. Thalamic lesions may be confined to one side, spread to both thalami or to the brainstem. Despite variations in anatomical manifestation, the molecular events dictating tumor spread and pathogenesis are not well defined. METHODS: A cohort of 294 specimens representing 207 subjects diagnosed gliomas with involvement of thalamus, were selected for methylation and whole genome sequencing using Illumina HIseq and EPIC bead array chips. Histological and clinical characterizations were correlated with molecular data to define unique pathways associated with tumor anatomical manifestation. RESULTS: Histological analysis indicated the presence of histone mutation in 46% of specimens. IHC based WHO grading indicated 44% (91/207) of patients with Grade 4, 21% (44/207) were Grade 3, 3% (6/207) were Grade 2, 22% (45/207) were Grade 1 and 10% were other types (e.g. ganglioglioma). Methylation studies conferred hypomethylation to be associated with H3 mutation status. Median age at diagnosis was 9.8 years and 56% of the cohort was male. The presence of the H3K27M mutation was associated with lower survival despite tumor grade. Molecular studies are underway to decipher specific pathways associated with LGG, HGG and TGs.

Pediatric brain cancers remain amongst the most lethal childhood cancers with over five decades of failed clinical trials. However, precision based clinical trials incorporating upfront biopsies, postmortem biobanking, along with technological advancements have resulted in a remarkable surge of omics and molecular based tumor classifications as well as a fundamental revision of WHO grading of pediatric brain tumors. However clinical translation of such molecular advancements has yet to impact a broad range of pediatric brain cancers. Major factors contributing to this lag of translation include, data fragmentation across institution, lack of data harmonization, data access, computational limitation, and the paucity of linked clinical and molecular data. To address these obstacles, we set to develop a unique data sharing platform for diffuse intrinsic pontine glioma (DIPG); one of the most devastating childhood brain caners. This initiative, named Project Open DIPG (POP DIPG) intends to consolidate and harmonize DIPG omics across all exiting and newly developed molecular data. Here we report that an international collaboration lead by Children’s Brain Tumor Tissue Consortium (CBTTC) has successfully launched POP DIPG housing the largest OMICs resource for DIPG and midline gliomas to date. Specifically POP DIPG houses the following molecular data: whole genome, whole exome, transcriptome, proteome, methylome of 850 unique subjects, over 1000 specimens and 20 avatar models of the disease. Harmonized and analyzed data are available through PedCBioportal providing the ease of access, rapid queries and hypothesis driven inquiries. Access to raw files is also granted though CBTTC to investigators with defined projects and hypotheses. With the expansion of POP DIPG, preclinical drug testing will be incorporated to accelerate discovery and more importantly to facilitate molecular-driven precision clinical trials. Open DIPG initiative will serve as a model for other challenging childhood cancers by bridging the current gap in bench to bedside translation.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an infiltrative, inoperable midline tumor with devastating outcomes. We have recently completed a prospective clinical trial for children and young adults with newly diagnosed DIPG that included clinical whole exome sequencing (WES) and RNA sequencing (RNAseq) of tumors and development of a personalized treatment plan. In this study, we retrospectively expanded sequencing efforts to include whole genome sequencing (WGS) and methylation profiling of paired tumor/normal (T/N) samples as well as longitudinal tumor specimens. We sought to perform a pan-omics analysis to determine which sequencing platform combination could most benefit patient outcomes in future precision medicine DIPG trials. METHODS: Thirty-three DIPG patients underwent successful biopsy in PNOC003. Clinical WES/exome panel sequencing and RNAseq was generated for 30 primary tumors and 2 progression tumors. We performed research-based WGS and methylation profiling of all tumors to do a pan-omics analysis. Cross-platform analysis was performed to compare WGS and WES and define additional mutations. RESULTS: In addition to WES and RNAseq, we performed WGS on all 33 primary T/N pairs, 2 progression samples, and 3 autopsy samples. Methylation profiling was done on 30 primary, 1 recurrent and 6 autopsy tumor samples. WGS identified all the actionable coding alterations called by WES and further identified non-coding variants of potential clinical significance. A pan-omics analysis spanning WGS, WES, RNAseq and methylation data is being performed. Tumor evolution was demonstrated by longitudinal comparison of genetic alterations across primary, recurrent and autopsy tumor samples to highlight tumor-intrinsic versus treatment-driven alterations in DIPGs. DISCUSSION: The ongoing pan-omic analysis from PNOC003 has begun informing the next generation of molecularly-driven DIPG clinical trials by suggesting inclusion of WGS in clinical sequencing panels as well as offering additional insights into the diverse mutational landscape of DIPGs.