Barbara S. Paugh

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.

Integrated Molecular Genetic Profiling of Pediatric High-Grade Gliomas Reveals Key Differences With the Adult Disease

PURPOSE To define copy number alterations and gene expression signatures underlying pediatric high-grade glioma (HGG). PATIENTS AND METHODS We conducted a high-resolution analysis of genomic imbalances in 78 de novo pediatric HGGs, including seven diffuse intrinsic pontine gliomas, and 10 HGGs arising in children who received cranial irradiation for a previous cancer using single nucleotide polymorphism microarray analysis. Gene expression was analyzed with gene expression microarrays for 53 tumors. Results were compared with publicly available data from adult tumors. RESULTS Significant differences in copy number alterations distinguish childhood and adult glioblastoma. PDGFRA was the predominant target of focal amplification in childhood HGG, including diffuse intrinsic pontine gliomas, and gene expression analyses supported an important role for deregulated PDGFRalpha signaling in pediatric HGG. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Pediatric and adult glioblastomas were clearly distinguished by frequent gain of chromosome 1q (30% v 9%, respectively) and lower frequency of chromosome 7 gain (13% v 74%, respectively) and 10q loss (35% v 80%, respectively). PDGFRA amplification and 1q gain occurred at significantly higher frequency in irradiation-induced tumors, suggesting that these are initiating events in childhood gliomagenesis. A subset of pediatric HGGs showed minimal copy number changes. CONCLUSION Integrated molecular profiling showed substantial differences in the molecular features underlying pediatric and adult HGG, indicating that findings in adult tumors cannot be simply extrapolated to younger patients. PDGFRalpha may be a useful target for pediatric HGG, including diffuse pontine gliomas.

The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma.

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.

Genome-Wide Analyses Identify Recurrent Amplifications of Receptor Tyrosine Kinases and Cell-Cycle Regulatory Genes in Diffuse Intrinsic Pontine Glioma

PURPOSE Long-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG. PATIENTS AND METHODS Single-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas. RESULTS Frequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase-Ras-phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar. CONCLUSION DIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG.

Targeted Therapy for BRAFV600E Malignant Astrocytoma

Purpose: Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAFV600E) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAFV600E in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAFV600E and wild-type BRAF MA. Experimental Design: BRAFV600E mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAFV600E MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo. Results: BRAFV600E mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAFV600E was identified in four instances. Using the BRAFV600E-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAFV600E mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. Conclusions: Our results indicate a 10% incidence of activating BRAFV600E among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAFV600E-specific inhibitors for treating BRAFV600E MA patients. Clin Cancer Res; 17(24); 7595–604. ©2011 AACR.

Novel Oncogenic PDGFRA Mutations in Pediatric High-Grade Gliomas

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.

Abstract PR03: The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma

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 Children9s 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.