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XLS file, 11K, PDGFRA sequencing primers.

XLS file, 10K, Histopathology of PDGFRa-driven tumors.

PDF file, 3383K, Figure-S1: Missense mutation resulting in alternative splicing and RT-PCR screens for KDR-PDGFRA fusion and PDGFRA ; Figure-S2: Analysis of PDGFR� downstream effectors ; Figure-S3: Mutant PDGFR� is sensitive to small molecule inhibitors ; Figure-S4: Dose-dependent sensitivity to crenolanib ; Figure-S5: Crenolanib and desatinib induce G0/G1 arrest ; Figure-S6: Signaling in PDGFRa-driven HGGs ; Figure-S7: PDGFR�-driven murine HGGs show a range of gene expression signatures.

Patients who undergo surgery for non-small cell lung cancer are at relatively high risk of local/regional recurrence, especially those with involved mediastinal lymph nodes. Postoperative radiation therapy (PORT) has been shown to decrease the risk of local/regional recurrence but its effect on overall survival remains unclear due to the competing risks of distant metastases and treatment-related morbidity and mortality. The randomized LungART study sought to examine the benefit of PORT, using modern techniques and doses, in patients with mediastinal lymph node involvement (N2) receiving adjuvant chemotherapy. Preliminary results show an improvement in local/regional control but no improvement in overall survival. While many guidelines continue to recommend PORT in the setting of positive margins or mediastinal lymph node involvement, these recommendations may change based on the final results of the LungART trial.

Diffuse intrinsic pontine glioma (DIPG) is most commonly diagnosed based on imaging criteria, with biopsy often reserved for pontine tumors with imaging features not typical for DIPG (atypical DIPG, ‘aDIPG’). The histopathologic and molecular spectra of the clinical entity aDIPG remain to be studied systematically. In this study, thirty-three patients with newly diagnosed pontine-centered tumors with imaging inconsistent with DIPG for whom a pathologic diagnosis was subsequently obtained were included. Neoplasms were characterized by routine histology, immunohistochemistry, interphase fluorescence in situ hybridization, Sanger and next-generation DNA/RNA sequencing, and genome-wide DNA methylome profiling. Clinicopathologic features and survival outcomes were analyzed and compared to those of a contemporary cohort with imaging features consistent with DIPG (typical DIPG, ‘tDIPG’). Blinded retrospective neuroimaging review assessed the consistency of the initial imaging-based diagnosis and correlation with histopathology. WHO grade II-IV infiltrating gliomas were observed in 54.6% of the cases; the remaining were low-grade gliomas/glioneuronal tumors or CNS embryonal tumors. Histone H3 K27M mutation, identified in 36% of the cases, was the major prognostic determinant. H3 K27M–mutant aDIPG and H3 K27M–mutant tDIPG had similar methylome profiles but clustered separately from diffuse midline gliomas of the diencephalon and spinal cord. In the aDIPG cohort, clinicoradiographic features did not differ by H3 status, yet significant differences in clinical and imaging features were observed between aDIPG without H3 K27M mutation and tDIPG. Neuroimaging review revealed discordance between the classification of aDIPG and tDIPG and did not correlate with the histology of glial/glioneuronal tumors or tumor grade. One patient (3.1%) developed persistent neurologic deficits after surgery; there were no surgery-related deaths. Our study demonstrates that surgical sampling of aDIPG is well-tolerated and provides significant diagnostic, therapeutic, and prognostic implications, and that neuroimaging alone is insufficient to distinguish aDIPG from tDIPG. H3 K27M-mutant aDIPG is epigenetically and clinically similar to H3 K27M-mutant tDIPG.

High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT downstream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics integrative approaches, and providing novel tumor vulnerabilities., Multi-omic profiling is a powerful approach to dissecting molecular mechanisms in disease. Here the authors generate whole proteome, phosphoproteome and transcriptome profiles from two mouse models of high-grade glioma driven by different oncogenes, and validate identified master regulators with a CRISPR screen.

Diffuse intrinsic pontine glioma (DIPG) is predominantly diagnosed clinically, with biopsy often reserved for cases with atypical imaging features and/or symptoms. Outcome data and molecular profiling of DIPG have been limited almost entirely to tumors with typical features. We examined the clinicopathologic characteristics and outcomes of 28 patients with pontine-centered lesions who underwent diagnostic biopsy at our institution from 2003 to 2018 because of atypical MRI findings. The median age at diagnosis was 5.2 years (range, 0.7–17.1), and the median symptom duration pre-diagnosis was 1 month (range, 0–24). Common atypical features included pontine asymmetry, tumor extension into the medulla and cerebellar peduncles, and exophytism. Four patients (14.3%) developed transient neurologic symptoms; one (3.6%) experienced a new, permanent cranial neuropathy; and no biopsy-related deaths occurred. Sixteen of the 28 tumors (57%) were confirmed histologically as DIPGs (WHO grade II–IV gliomas; “atypical DIPG”). Of these 16 cases, H3 K27M status was assessed through immunohistochemistry in 14 (88%) and nine were immunopositive (64.3%). The remainder (“atypical non-DIPGs”) were pilocytic astrocytomas (58.3%), gangliogliomas (8.3%), CNS embryonal tumors, NOS (16.7%), or C19MC-altered embryonal tumors with multilayered rosettes (16.7%). With a median follow-up of 28.6 months (range, 3.0 – 173.2), 2-year overall survival (OS) was 72.4%, 83.3%, and 62.2% for the total cohort, atypical non-DIPGs, and atypical DIPGs, respectively (P=NS for all). However, OS with H3 K27M–mutant atypical DIPG was inferior to that with H3 WT atypical DIPG (P=0.003). Compared to OS in a contemporary typical DIPG cohort (N=100), OS was significantly longer for the total atypical cohort, atypical non-DIPGs, and atypical DIPGs (P

A greater understanding of the unique drivers of pediatric high-grade gliomas (pHGG) is needed as the two-year survival rate is only 10–30%. We recently discovered the presence of structural variants in 47% of pHGG. Recurrent fusions were found involving neurotrophin receptors (NTRK) in 7% of all pHGG and 40% of non-brainstem HGG in infants. One chimeric gene we identified encoded the ETS transcription factor, ETV6, fused to the kinase domain of NTRK3 causing ligand-independent kinase activity. ETV6-NTRK3 has been identified in other neoplasms including congenital-infantile fibrosarcoma and AML. To examine the role of ETV6-NTRK3 in glioma formation in vivo, we developed an implantation model. Cortical astrocytes with conditional loss of p53 in neural stem cells were harvested from mice at P2, transduced with a vector expressing ETV6-NTRK3, and were implanted into the cortex of adult mice. Even with a transduction efficiency of only 1.5–2%, all mice implanted with transduced astrocytes developed HGG with a median survival of 36 days. Next, to study ETV6-NTRK3 in the development of pediatric gliomas, we generated a spontaneous tumor model in which mice expressed a conditional knock-in ETV6-NTRK3 allele and conditional loss of p53 in primitive neural stem cells. Mice expressing ETV6-NTRK3 and null p53 showed rapid development and complete penetrance of high-grade glioma with a median survival of 18 days. Evidence of HGG development was readily detectable by P5, accurately recapitulating onset of pediatric gliomagenesis. Analysis showed ETV6-NTRK3 primary astrocytes had increased proliferation compared to control astrocytes and HGGs driven by ETV6-NTRK3 showed a high proliferative index, MAPK, PI3K and PLCγ activity. In vitro and in vivo models were employed to identify oncogenic targets downstream of NTRK fusions and for preclinical testing of TRK selective inhibitors.

Advances in understanding pediatric high-grade glioma (pHGG) genetics have revealed key differences between pediatric and adult high-grade gliomas (aHGGs), and have uncovered unique molecular drivers among subgroups within pHGG. The three core aHGG pathways, the receptor tyrosine kinase(RTK)/Ras/Phosphatidylinositide 3-kinase (PI3K), p53, and retinoblastoma (RB) networks, are also disrupted in pHGG, but they exhibit a different spectrum of effectors targeted by mutation. There are also similarities and differences in the genomic landscape of diffuse intrinsic pontine glioma (DIPG) and pediatric non-brainstem high-grade glioma (pNBS-HGG). In 2012, histone H3 mutations were identified in nearly 80% of DIPGs and ∼35% of pNBS-HGG. These were the first reports of histone mutations in human cancer, implicating novel biology in pediatric gliomagenesis. Additionally, DIPG and midline pNBS-HGG vary in the frequency and specific histone H3 amino acid substitution compared to pNBS-HGGs arising in the cerebral hemispheres, demonstrating a molecular difference among pHGG subgroups. The gene expression signatures as well as DNA methylation signatures of these tumors also carry distinctive signatures, reflecting a combination of the driving mutations and the developmental context from which they arise. These data collectively highlight unique selective pressures within the developing brainstem and solidify DIPG as a specific molecular and biological entity among pHGGs. Emerging studies continue to identify novel mutations that distinguish subgroups of pHGG. The molecular heterogeneity among pHGGs will undoubtedly have clinical implications moving forward. The discovery of unique oncogenic drivers is a critical first step in providing patients with appropriate, targeted therapies. Despite these insights, our vantage point has been largely limited to an in-depth analysis of protein coding sequences. Given the clear importance of histone mutations in pHGG, it will be interesting to see how aberrant epigenetic regulation contributes to tumorigenesis in the pediatric context. New mechanistic insights may allow for the identification of distinct vulnerabilities in this devastating spectrum of childhood tumors.

Pediatric high-grade glioma (HGG) is a devastating disease with a less than 20% survival rate 2 years after diagnosis. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs), by whole-genome, whole-exome and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPGs (32%), in addition to previously reported frequent somatic mutations in histone H3 genes, TP53 and ATRX, in both DIPGs and NBS-HGGs. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, NTRK2 and NTRK3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase-RAS-PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59% of pediatric HGGs, respectively, including in DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.

The outcome for children with high-grade gliomas (HGG) remains dismal, with a 2-year survival rate of only 10% to 30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in the brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet-derived growth factor receptor α (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic-activating mutations were identified in 14.4% (13 of 90) of nonbrainstem pediatric HGGs and 4.7% (2 of 43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. Forty percent of tumors with mutation showed concurrent amplification, whereas 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 nonbrainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG. Cancer Res; 73(20); 6219–29. ©2013 AACR.

High-grade Brainstem glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is thought to be a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including upregulation of paired box 3 (Pax3). In the neonatal mouse brain, while Pax3 expression is absent from the cerebral cortex, its expression marks a subset of brainstem progenitor cells, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by significantly shortening tumor latency (p = 0.02 by log rank test), and increasing tumor penetrance (from 25% to 80%, p = 0.0003 by Fisher's exact test), in a region-specific manner, while loss of Pax3 function in PDGF-B-driven;p53-deficient BSG significantly delays glioma formation (median survival of 38 versus 28.5 days, p = 0.0007 by log rank test). Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations and amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.

Diffuse high-grade gliomas (HGGs) of childhood are a spectrum of disease with devastatingly poor outcome. Despite similar histological features, pediatric HGGs arise from a broader distribution of anatomical locations when compared to adults, with approximately 50% arising in the brainstem as diffuse intrinsic pontine glioma (DIPG), a disease found almost exclusively in children. Recent genome-wide studies provided abundant evidence that unique selective pressures drive HGG in children compared to adults, identifying novel oncogenic mutations connecting tumorigenesis and chromatin regulation as well as developmental signaling pathways. The striking association between frequencies of specific mutations with spatiotemporal pattern of HGG formation in children further highlights context-dependent connections between developmental states and oncogenic drivers. Activating mutations in the BMP receptor ACVR1 are found exclusively in the youngest subgroup of DIPG patients, and not identified in HGGs outside the brainstem. Recurrent mutations in histone H3 establish a critical role for epigenetic regulation in disease pathogenesis of childhood HGG. H3K27M mutations occur in nearly 80% of DIPGs and more than half of HGGs arising in midline structures outside the brainstem such as the thalamus. In contrast, approximately 15% of HGGs arising in the cerebral cortex have an alternative H3 mutation, G34R/V, and are predominantly found in older adolescents and young adults. To study these mutations in the developing mammalian brain, we generated conditionally activated, epitope tagged H3f3a knock-in mice to express K27M, G34R or non-mutated H3.3 proteins from the endogenous H3f3a locus. We will present early analyses of these new oncohistone mouse models. Citation Format: Jon D. Larson, Andre B. Silveira, Lawryn H. Kasper, Alexander K. Diaz, Xiaoyan Zhu, Suzanne J. Baker. The unique genomic and epigenomic landscape of pediatric high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA15.

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with no effective cures. DIPG arises in the brainstem and is found almost exclusively in children. Mutations in canonical cancer signalling pathways including the RTK/RAS/PI3K pathway, the Tp53 pathway, and the CDK4/6 G1 checkpoint occur in DIPGs, targeting different effectors within these pathways and resulting in significant genetic heterogeneity between tumors. However, genome-wide studies provided abundant evidence that unique selective pressures drive HGG in children compared to adults, identifying novel oncogenic mutations connecting tumorigenesis and chromatin regulation as well as developmental signaling pathways. Recurrent mutations of the BMP receptor ACVR1/ALK2 are found in 25-30% of DIPGs, and are further restricted to the youngest patients, highlighting critical connections between development and gliomagenesis. The molecular hallmark of DIPGs is frequent recurrent somatic mutation in histone H3. Mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively, are found in nearly 80 percent of DIPGs. These recurrent mutations provide clear evidence of an important role for chromatin regulation in DIPG disease pathogenesis, and present a unifying molecular mechanism driving DIPG. We have evaluated the contribution of H3.3 K27M mutation to DIPG tumor growth in vitro to gain insights into molecular mechanisms of oncogenic activity, and to assess the potential of histone H3 mutation as a therapeutic sensitivity in this incurable brain tumor. Citation Format: Andre B. Silveira, Lawryn H. Kasper, Jon D. Larson, Xiaoyan Zhu, Alexander K. Diaz, Suzanne J. Baker. Oncogenic mechanisms in diffuse intrinsic pontine gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA26.

Pediatric high-grade glioma (HGG) remains a tremendous clinical challenge, with a two-year survival of less than 20%. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs) by whole-genome, whole-exome, and/or transcriptome sequencing. Somatic mutations in the bone morphogenetic protein (BMP) receptor ACVR1 occurred in 32% of DIPG, a finding exclusive to brainstem HGG. Structural variants generating fusion genes were found in 47% of pediatric HGG, with recurrent fusions involving the neurotrophin receptor genes NTRK1, 2, or 3 in 40% of infant NBS-HGGs and 5% of pediatric HGG overall. Multiple mutations targeted pathways involving histone modification or chromatin remodeling, cell cycle regulation and receptor tyrosine kinase/RAS/PI3K signaling, in both DIPG and NBS-HGGs at frequencies of greater than 39% in the entire cohort. The HGG mutation burden ranged from 2 non-silent mutations in an infant HGG to more than a million mutations in a tumor associated with germline mismatch repair deficiency. From these findings, we have established novel tumor models to better understand this devastating disease. This work provides new insight into the genetic events driving pediatric HGG tumorigenesis. This abstract is also presented as Poster B14. Citation Format: Alexander K. Diaz, Gang Wu, Barbara S. Paugh, Yongjin Li, Xiaoyan Zhu, Sherri Rankin, Chunxu Qu, Xiang Chen, Junyuan Zhang, John Easton, Michael Edmonson, Charles Lu, Panduka Nagahawatte, Erin Hedlund, Michael Rusch, Stanley Pounds, Tong Lin, Arzu Onar-Thomas, Robert Huether, Richard Kriwacki, Matthew Parker, Pankaj Gupta, Jared Becksfort, Lei Wei, Heather L. Mulder, Kristy Boggs, Bhavin Vadodaria, Donald Yergeau, Kerri Ochoa, Robert S. Fulton, Lucinda S. Fulton, Chris Jones, Alberto Broniscer, Cynthia Wetmore, Amar Gajjar, Li Ding, Elaine R. Mardis, Richard K. Wilson, James R. Downing, David W. Ellison, Jinghui Zhang, Suzanne J. Baker, For the St Jude Children's Research Hospital – Washington University Pediatric Cancer Genome Project. The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR03.

High-grade gliomas (HGG) account for twenty percent of primary brain tumors in children and have a devastating outcome with a 2-year survival rate of less than 30%. While adult HGG arise frequently in the cerebral cortex, pediatric HGG can form in cerebral cortex as well as midline regions including thalamus and brainstem, and are genetically distinct from adult tumors. Brainstem HGGs, referred to as diffuse intrinsic pontine glioma (DIPG), are mostly exclusive to children and confer a dismal less than 10% 2-year survival rate. Recent genome-wide sequence analysis of pediatric DIPG and non-brainstem HGG revealed a predominance of mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively. These mutations were found in 78% of DIPG patients and 22% of non-brainstem pediatric HGG. An additional mutually exclusive somatic heterozygous H3F3A mutation encoding histone H3.3 G34R was identified in 14% of non-brainstem HGG. We established xenografts from human non-brainstem HGGs carrying wild-type H3 or the H3.3 G34R mutant, and DIPGs with H3.3 or H3.1 K27M mutations. H3 K27M mutant xenografts showed a marked reduction in H3K27 trimethylation (H3K27me3), while tumors with wild-type or H3.3 G34R mutations retain H3K27me3 as demonstrated by immunohistochemistry and Western blot analyses. To determine if this was a dominant effect of K27M mutation or a developmental signature of DIPG we transduced primary astrocyte cultures derived from p53-deficient neonatal mouse cortex or brainstem with retrovirus expressing wild-type, K27M, K27A or G34R H3 mutant variants. Low-level expression of exogenous H3.3 K27M compared to endogenous H3 was sufficient to recapitulate prominent H3K27me3 reduction observed in the xenografts. Furthermore, we evaluated correlation between H3 mutation and H3K27me3 status in a large cohort of primary pediatric HGG including DIPG. From this we identified a significant relationship between H3 K27M mutation and H3K27me3 loss in DIPG. However, we also observed rare wild-type H3 DIPG cases exhibiting reduced H3K27me3 indicating a potential alternative mechanism for this methylation state. In addition, we evaluated the association of H3K27me3 and K27M mutations with genome-wide DNA methylation patterns, and gene expression signatures. Together, these results implicate histone H3 K27M and G34R mutations as unique, dominant genetic drivers in distinct subtypes of pediatric HGG. Current efforts focus on delineating the divergent pathological roles of H3 K27M and G34R mutations during DIPG and non-brainstem HGG tumorigenesis. Citation Format: Jon D. Larson, Troy A. McEachron, Chunxu Qu, Xiaoyan Zhu, Alexander K. Diaz, David W. Ellison, Brent A. Orr, Suzanne J. Baker. Variant histone H3 mutations associate with histone modification, DNA methylation, and gene expression changes in pediatric high-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A15.