Ruth G. Tatevossian

Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.

C11orf95 – RELA fusions drive oncogenic NF-κB signalling in ependymoma

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95–RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95–RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95–RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549–BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto‐inhibitory domains of BRAF and RAF1, which are replaced in‐frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low‐grade gliomas. Copyright

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.

MAPK pathway activation and the origins of pediatric low-grade astrocytomas

Low‐grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment. J. Cell. Physiol. 222: 509–514, 2010.

The Genomic Landscape of Childhood and Adolescent Melanoma

Despite remarkable advances in the genomic characterization of adult melanoma, the molecular pathogenesis of pediatric melanoma remains largely unknown. We analyzed 15 conventional melanomas (CMs), 3 melanomas arising in congenital nevi (CNMs), and 5 spitzoid melanomas (SMs), using various platforms, including whole genome or exome sequencing, the molecular inversion probe assay, and/or targeted sequencing. CMs demonstrated a high burden of somatic single-nucleotide variations (SNVs), with each case containing a TERT promoter (TERT-p) mutation, 13/15 containing an activating BRAF V600 mutation, and >80% of the identified SNVs consistent with UV damage. In contrast, the three CNMs contained an activating NRAS Q61 mutation and no TERT-p mutations. SMs were characterized by chromosomal rearrangements resulting in activated kinase signaling in 40%, and an absence of TERT-p mutations, except for the one SM that succumbed to hematogenous metastasis. We conclude that pediatric CM has a very similar UV-induced mutational spectrum to that found in the adult counterpart, emphasizing the need to promote sun protection practices in early life and to improve access to therapeutic agents being explored in adults in young patients. In contrast, the pathogenesis of CNM appears to be distinct. TERT-p mutations may identify the rare subset of spitzoid melanocytic lesions prone to disseminate.

RAF gene fusions are specific to pilocytic astrocytoma in a broad paediatric brain tumour cohort

Brain tumours are the most common solid tumour in children and are the primary cause of cancer-related death in children and young adults [4, 6]. The most prevalent childhood brain tumours are low-grade gliomas, specifically pilocytic astrocytomas (PAs, WHO Grade I) [1]. PAs are slow-growing tumours which are often cystic, and may occur sporadically or in association with the genetic disorder Neurofibromatosis type 1. Several recent studies including our own have identified novel KIAA1549–BRAF and SRGAP3–RAF1 gene fusions in the majority of PAs tested [3, 7, 8, 12]. In these fusions, the N-terminal autoinhibitory domains of the RAF proteins are replaced by those of KIAA1549 or SRGAP3, resulting in constitutive activation of the ERK/MAPK pathway. A recent study has suggested that the KIAA1549–BRAF fusion is more common in PAs originating in the cerebellum [5]. In low-grade glioma without RAF gene fusions there is increasing evidence for activation of the ERK/MAPK pathway through alternative mechanisms, such as point mutation of KRAS or BRAF [2, 11, 13]. Despite the high frequency of RAF gene fusions in PAs, they have not been investigated in other types of paediatric brain tumours. In this study, we screened a new cohort of 74 paediatric brain tumours, with a range of different pathologies, for all known KIAA1549–BRAF and SRGAP3–RAF1 fusion variants. Access to tumours and clinical data was in accordance with Local Research Ethics Committee (LREC) regulations: Great Ormond Street Hospital LREC reference number 05/Q0508/153. Tumours were classified by diagnostic criteria defined by the World Health Organization (WHO) [10]. Total RNA was extracted from fresh frozen tissue samples using the miRNeasy mini kit (Qiagen, Crawley, UK) and reverse transcribed using the SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA). KIAA1549–BRAF fusions were detected using previously described primers and techniques [3]. The primers used for detecting SRGAP3–RAF1 fusions were 50-TGG CAGTAACCTCATCACCA-30 (located in SRGAP3 exon 10) and 50-GGTTGGGTCGACAACCTTTA-30 (located in RAF1 exon 11). All fusions identified by PCR were confirmed by direct sequencing on a 3100 Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0693-y) contains supplementary material, which is available to authorized users.

RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology

Gene fusions involving members of the RAF family of protein kinases have recently been identified as characteristic aberrations of low-grade astrocytomas, the most common tumors of the central nervous system in children. While it has been shown that these fusions cause constitutive activation of the ERK/MAPK pathway, very little is known about their formation. Here, we present a detailed analysis of RAF gene fusion breakpoints from a well-characterized cohort of 43 low-grade astrocytomas. Our findings show that the rearrangements that generate these RAF gene fusions may be simple or complex and that both inserted nucleotides and microhomology are common at the DNA breakpoints. Furthermore, we identify novel enrichment of microhomologous sequences in the regions immediately flanking the breakpoints. We thus provide evidence that the tandem duplications responsible for these fusions are generated by microhomology-mediated break-induced replication (MMBIR). Although MMBIR has previously been implicated in the pathogenesis of other diseases and the evolution of eukaryotic genomes, we demonstrate here that the proposed details of MMBIR are consistent with a recurrent rearrangement in cancer. Our analysis of repetitive elements, Z-DNA and sequence motifs in the fusion partners identified significant enrichment of the human minisatellite conserved sequence/χ-like element at one side of the breakpoint. Therefore, in addition to furthering our understanding of low-grade astrocytomas, this study provides insights into the molecular mechanistic details of MMBIR and the sequence of events that occur in the formation of genomic rearrangements.

Clinical, radiological, histological and molecular characteristics of paediatric epithelioid glioblastoma

A few case series in adults have described the characteristics of epithelioid glioblastoma (e‐GB), one of the rarest variants of this cancer. We evaluated clinical, radiological, histological and molecular characteristics in the largest series to date of paediatric e‐GB.

Molecular Characterization of Choroid Plexus Tumors Reveals Novel Clinically Relevant Subgroups

Purpose: To investigate molecular alterations in choroid plexus tumors (CPT) using a genome-wide high-throughput approach to identify diagnostic and prognostic signatures that will refine tumor stratification and guide therapeutic options. Experimental Design: One hundred CPTs were obtained from a multi-institutional tissue and clinical database. Copy-number (CN), DNA methylation, and gene expression signatures were assessed for 74, 36, and 40 samples, respectively. Molecular subgroups were correlated with clinical parameters and outcomes. Results: Unique molecular signatures distinguished choroid plexus carcinomas (CPC) from choroid plexus papillomas (CPP) and atypical choroid plexus papillomas (aCPP); however, no significantly distinct molecular alterations between CPPs and aCPPs were observed. Allele-specific CN analysis of CPCs revealed two novel subgroups according to DNA content: hypodiploid and hyperdiploid CPCs. Hyperdiploid CPCs exhibited recurrent acquired uniparental disomy events. Somatic mutations in TP53 were observed in 60% of CPCs. Investigating the number of mutated copies of p53 per sample revealed a high-risk group of patients with CPC carrying two copies of mutant p53, who exhibited poor 5-year event-free (EFS) and overall survival (OS) compared with patients with CPC carrying one copy of mutant p53 (OS: 14.3%, 95% confidence interval, 0.71%–46.5% vs. 66.7%, 28.2%–87.8%, respectively, P = 0.04; EFS: 0% vs. 44.4%, 13.6%–71.9%, respectively, P = 0.03). CPPs and aCPPs exhibited favorable survival. Discussion: Our data demonstrate that differences in CN, gene expression, and DNA methylation signatures distinguish CPCs from CPPs and aCPPs; however, molecular similarities among the papillomas suggest that these two histologic subgroups are indeed a single molecular entity. A greater number of copies of mutated TP53 were significantly associated to increased tumor aggressiveness and a worse survival outcome in CPCs. Collectively, these findings will facilitate stratified approaches to the clinical management of CPTs. Clin Cancer Res; 21(1); 184–92. ©2014 AACR.