Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma

Abstract

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. 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 new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

Figure 1. Novel BRAF alterations in pilocytic astrocytoma

a, Schematic representation of the RNF130:BRAF fusion gene in ICGC_PA112 resulting from a translocation between chromosomes 5 and 7. A similar fusion was observed in ICGC_PA96. The cDNA sequence at the fusion breakpoint and resulting exon and protein structures are indicated. A reciprocal fusion between RUN and FYVE domain containing 1 (RUFY1) and transmembrane protein 178B (TMEM178B) on the derivative chromosome 5 in ICGC_PA112 was also found to be expressed by RNA-seq analysis (not shown). RPM, reads per million; KD, kinase domain. b, Computational modeling of two BRAF monomers (light/dark grey) with a VLR insertion (blue/magenta) between Arg506 and Lys507 (green), as identified in ICGC_PA65. The PDB structure 4E26 was used as a template. Val600, a mutational hotspot, is shown in yellow. A novel dimer interface is formed between the protomers, with hydrogen bonds from the new arginine side chains (dashed lines) and a hydrophobic interaction between the leucine side chains (magenta). c, Western blot analysis of NIH3T3 cells transfected with empty vector (EV), BRAF^WT, BRAF^V600E and BRAF^insVLR. The newly identified insVLR mutant results in elevation of ERK1/2 phosphorylation (pERK1/2) at a similar level to that seen with the known oncogenic V600E form. d, Co-immunoprecipation assay with pull-down of HA-tagged BRAF^insVLR, demonstrating that this novel mutant forms homodimers with co-transfected AU1-tagged insVLR, but does not appear to form a strong heterodimer with wild-type BRAF.

Figure 2. NTRK2 is a new gene fusion target in pilocytic astrocytoma

Schematic representation of the QKI:NTRK2 fusion gene in ICGC_PA159 resulting from a translocation between chromosomes 6 and 9. A similar fusion was observed in ICGC_PA82. The cDNA sequence at the fusion breakpoint and resulting exon and protein structures are indicated. RPM, reads per million; KD, kinase domain; QUA1, Qua1 dimerization domain.

Figure 3. FGF pathway signaling molecules are recurrently altered in pilocytic astrocytoma

a, Schematic of the domain structure of FGFR1, indicating the position and frequency of the hotspot mutations in pilocytic astrocytomas sequenced in the present study (including replication cases). b, Gene expression data for FGF2 indicating significantly elevated expression in pilocytic astrocytomas (red) compared with other astrocytic tumors (blue), normal cerebellum (black) or other normal tissues (green); P <0.001, two-sided t-test. The pilocytic astrocytomas with expression data which harbor FGFR1 alterations (four mutants plus FGFR1-ITD) are highlighted. c, Schematic of an additional alteration in FGFR1 identified in ICGC_PA89, comprising an internal tandem duplication (ITD) of part of intron 10, exons 11–17 and part of exon 18. The duplicated amino acids are aa478–820 (numbered according to the alpha A1 isoform), with an additional 40 amino acid linker sequence encoded by part of intron 10. The whole kinase domain is therefore duplicated in the resulting predicted protein (TK1′ and TK2′). d, Schematic of the structure of PTPN11 (SHP-2), indicating the position and frequency of mutations in pilocytic astrocytomas sequenced in the present study. e, Gene expression data for PTPN11 indicating significantly elevated expression in pilocytic astrocytomas compared with other groups, as in (b); P <0.001, two-sided t-test. The pilocytic astrocytomas with expression data which harbor FGFR1 alterations (four mutants plus FGFR1-ITD) are highlighted. Ig, immunoglobulin-like domain; TM, transmembrane domain; TK, tyrosine kinase domain; SH2, src homology 2 domain; PTP, protein tyrosine phosphatase domain; DA, WHO Grade II diffuse astrocytoma; AA, anaplastic astrocytoma; K27/G34/IDH1 GBM, glioblastoma carrying a mutation at K27 or G34 in H3F3A or in IDH1; ped., pediatric; CBM, cerebellum, CNS, central nervous system.

Figure 4. Summary of MAPK pathway alterations in pilocytic astrocytoma

An overview of MAPK pathway alterations identified in the 96 whole-genome sequencing cases included in the present study, indicating the mutual exclusivity of the majority of these hits (with the exception of FGFR1 and PTPN11); P <0.0001, permutation test on 10,000 iterations. Each column represents one tumor sample. Red filled boxes indicate that a given alteration is present in this sample. The blue filled box represents FGFR1-ITD rather than a point mutation; the green box indicates a BRAF^E451D mutation in a case with a KIAA1549:BRAF fusion (unknown functional significance, but included in the exclusivity testing); the black/red boxes indicate that the NF1 alterations comprise one germline and one somatic hit per case.