Pilocytic astrocytoma, the most common childhood brain tumor1, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations2. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression3 and often becoming a chronic disease with substantial morbidities4. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n=73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and novel NTRK2 fusion genes in non-cerebellar tumors. New BRAF activating changes were also observed. MAPK pathway alterations affected 100% of tumors analyzed, with no other significant mutations, indicating pilocytic astrocytoma as predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in NF15. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.
Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.
Pilocytic astrocytomas (PAs) are the most common brain tumors in pediatric patients and can cause significant morbidity, including chronic neurological deficiencies. They are characterized by activating alterations in the mitogen-activated protein kinase pathway, but little else is known about their development. To map the global DNA methylation profiles of these tumors, we analyzed 62 PAs and 7 normal cerebellum samples using Illumina 450K microarrays. These data revealed two subgroups of PA that separate according to tumor location (infratentorial versus supratentorial), and identified key neural developmental genes that are differentially methylated between the two groups, including NR2E1 and EN2. Integration with transcriptome microarray data highlighted significant expression differences, which were unexpectedly associated with a strong positive correlation between methylation and expression. Differentially methylated probes were often identified within the gene body and/or regions up- or downstream of the gene, rather than at the transcription start site. We also identified a large number of differentially methylated genes between cerebellar PAs and normal cerebellum, which were again enriched for developmental genes. In addition, we found a significant association between differentially methylated genes and SUZ12 binding sites, indicating potential disruption of the polycomb repressor complex 2 (PRC2). Taken together, these data suggest that PA from different locations in the brain may arise from region-specific cells of origin, and highlight the potential disruption of key developmental regulators during tumorigenesis. These findings have implications for future basic research and clinical trials, as therapeutic targets and drug sensitivity may differ according to tumor location.
Purpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS) through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnormalities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain neoplasm in children. We investigated whether OIS is induced in PA.Experimental Design: We tested expression of established senescence markers in three independent cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized astrocytes and fetal astrocytes.Results: Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic b-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA samples (46 of 52; 88.5%). Overexpression of a number of senescence-associated genes [CDKN2A (p16), CDKN1A (p21), CEBPB, GADD45A, and IGFBP7] was shown at the mRNA level in two independent PA tumor series. In vitro, sustained activation of wild-type or mutant BRAF induced OIS in both astrocytic cell lines. Loss of p16 INK4a in immortalized astrocytes abrogated OIS, indicative of the role of this pathway in mediating this phenomenon in astrocytes. OIS is a mechanism of tumor suppression that restricts the progression of benign tumors. We show that it is triggered in PAs through p16 INK4a pathway induction following aberrant MAPK activation. Conclusions: OIS may account for the slow growth pattern in PA, the lack of progression to higher-grade astrocytomas,andthe highoverall survival ofaffected patients.Clin CancerRes; 17(14); 4650-60.'2011 AACR.
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