ARTICLE doi:10.1038/nature11327 Subgroup-specific structural variation across 1,000 medulloblastoma genomes A list of authors and their affiliations appears at the end of the paper Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson’s disease, which is exquisitely restricted to Group 4a. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-b signalling in Group 3, and NF-kB signalling in Group 4, suggest future avenues for rational, targeted therapy. Brain tumours are the most common cause of childhood oncological Twenty-eight regions of recurrent high-level amplification (copy death, and medulloblastoma is the most common malignant paediatric number $ 5) were identified (Fig. 1d and Supplementary Table 7). brain tumour. Current medulloblastoma therapy including surgical The most prevalent amplifications affected members of the MYC resection, whole-brain and spinal cord radiation, and aggressive family with MYCN predominantly amplified in SHH and Group 4, chemotherapy supplemented by bone marrow transplant yields five- MYC in Group 3, and MYCL1 in SHH medulloblastomas. Multiple year survival rates of 60–70%1. Survivors are often left with significant genes/regions were exclusively amplified in SHH, including GLI2, neurological, intellectual and physical disabilities secondary to the MYCL1, PPM1D, YAP1 and MDM4 (Fig. 1d). Recurrent homozygous effects of these nonspecific cytotoxic therapies on the developing brain2. deletions were exceedingly rare, with only 15 detected across 1,087 Recent evidence suggests that medulloblastoma actually comprises tumours (Fig. 1e). Homozygous deletions targeting known tumour multiple molecularly distinct entities whose clinical and genetic dif- suppressors PTEN, PTCH1 and CDKN2A/B were the most common, ferences may require separate therapeutic strategies3–6. Four principal all enriched in SHH cases (Fig. 1e and Supplementary Table 7). Novel subgroups of medulloblastoma have been identified: WNT, SHH, homozygous deletions included KDM6A, a histone-lysine demethylase Group 3 and Group 4 (ref. 7), and there is preliminary evidence for deleted in Group 4. A custom nanoString CodeSet was used to verify 24 clinically significant subdivisions of the subgroups3,7,8. Rational, significant regions of gain across 192 MAGIC cases, resulting in a targeted therapies based on genetics are not currently in use for verification rate of 90.9% (Supplementary Fig. 5). We conclude that medulloblastoma, although inhibitors of the Sonic Hedgehog SCNAs in medulloblastoma are common, and are predominantly pathway protein Smoothened have shown early promise9. Actionable subgroup-enriched. targets for WNT, Group 3 and Group 4 tumours have not been identified4,10. Sanger sequencing of 22 medulloblastoma exomes Subgroup-specific SCNAs in medulloblastoma revealed on average only 8 single nucleotide variants (SNVs) per WNT medulloblastoma genomes are impoverished of recurrent focal tumour11. Some SNVs were subgroup-restricted (PTCH1, CTNNB1), regions of SCNA, exhibiting no significant regions of deletion and whereas others occurred across subgroups (TP53, MLL2). We pro- only a small subset of focal gains found at comparable frequencies in posed that the observed intertumoural heterogeneity might have non-WNT tumours (Supplementary Figs 4, 6 and Supplementary underpowered prior attempts to discover targets for rational therapy. Table 8). CTNNB1 mutational screening confirmed canonical exon The Medulloblastoma Advanced Genomics International 3 mutations in63 out of 71 (88.7%) WNT tumours, whereas monosomy Consortium (MAGIC) consisting of scientists and physicians from 6 was detected in 58 out of 76 (76.3%) (Supplementary Fig. 6; 46 cities across the globe gathered more than 1,200 medulloblastomas Supplementary Table 9). Four WNT tumours (4/71; 5.6%) had neither which were studied by SNP arrays (n 5 1,239; Fig. 1a, Supplementary CTNNB1 mutation nor monosomy 6, but maintained typical WNT Fig. 1 and Supplementary Tables 1–3). Medulloblastoma subgroup expression signatures. Given the size of our cohort and the resolution affiliation of 827 cases was determined using a custom nanoString- of the platform, we conclude that there are no frequent, targetable based RNA assay (Supplementary Fig. 2)12. Disparate patterns of SCNAs for WNT medulloblastoma. broad cytogenetic gain and loss were observed across the subgroups SHH tumours exhibit multiple significant focal SCNAs (Fig. 2a, (Fig. 1b and Supplementary Figs 3, 7, 8, 10 and 11). Analysis of the Supplementary Figs 12, 15, 16 and Supplementary Tables 10 and 11). entire cohort using GISTIC2 (ref. 13) to discover significant ‘driver’ SHH enriched/restricted SCNAs included amplification of GLI2 and events delineated 62 regions of recurrent SCNA (Fig. 1c, deletion of PTCH1 (Fig. 2a, e, f)10. MYCN and CCND2 were among Supplementary Fig. 4 and Supplementary Tables 4 and 5); analysis the most frequently amplified genes in SHH (Supplementary by subgroup increased sensitivity such that 110 candidate ‘driver’ Table 6), but were also altered in non-SHH cases. Genes upregulated SCNAs were identified, most of which are subgroup-enriched in SHH tumours (that is, SHH signature genes) are significantly over- (Fig. 1c–e and Supplementary Table 6). represented among the genes focally amplified in SHH tumours 2AUGUST2012|VOL488|NATURE|49 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE WNT SHH Group 3 Group 4 abcWNT SHH Group 3 Group 4 Loss frequency n n n n ( = 76) ( = 266) ( = 168) ( = 317) 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 1.0 0.5 0 Pan-cohort Analysis by analysis subgroup 1 1 2 100 2 3 3 4 5 80 4 6 7 60 5 8 6 9 10 40 7 11 8 12 9 13 Number of events 20 14 10 Chromosome 15 11 16 0 12 17 P = 0.02402 13 Chromosome arm (p,q) 18 14 19 Subgroup 15 20 16 WNT 17 21 18 SHH 19 22 20 Group 3 21 Group 4 22 Log copy number ratio 011.00.5 0.50 .00 0.5 1.0 1.00.50 Non-specific 2 Gain frequency –1.0 0.0 1.0 Gain Significant gain Deletion Significant deletion (CN 1.0) (CN 2.0) (CN 4.0) d Recurrent high-level amplifications Freq. e Recurrent homozygous deletions Freq. MYCN (2) (3.5%) PTEN (5) MYC (1) (2.9%) (0.7%) GLI2 (1) (1.3%) PTCH1 (5) CDK6 (3) ACVR2B (4) (1.1%) CDKN2A/B (8) (0.6%) MYCL1 (6) (0.6%) PPM1D (3) KDM6A (N/A) CCND2 (0.5%) (2) CD99 YAP1 (25) (N/A) (0.3%) ACVR2A (1) (0.4%) PPP2R3B PTBP1 (11) (N/A) SMC1A (N/A) EHMT1 (N/A) TNFRSF13B (2) MDM4 (1) (0.3%) IKBKAP (N/A) KIT (1) CDK4 (13) TSC1 (4) SMPX (N/A) PIM1 (N/A) RBMS3 (N/A) GPR87 (N/A) Subgroup Subgroup IGF1R (6) SOX12 (31) (0.2%) LMO4 (N/A) WNT WNT MAP2K4 TP53 (19) (N/A) SHH (0.2%) SHH MIR17HG (N/A) DUSP21 TGFBR1 (4) Group 3 (N/A) Group 3 PHF2 (N/A) Group 4 NLGN4X Group 4 KAT6A (N/A) (N/A) Non-specific Non-specific DLC1 (119) MAP2K4 (N/A) ZIK1 (N/A) 0 5 10 15 20 25 30 35 40 0123456789 No. of cases No. of cases Figure 1 | Genomic heterogeneity of medulloblastoma subgroups. a,The (d, segmented copy number (CN) $ 5) and homozygous deletions medulloblastoma genome classified by subgroup. b, Frequency and significance (e, segmented CN # 0.7) in medulloblastoma. The number of genes mapping to (Q value # 0.1) of broad cytogenetic events across medulloblastoma subgroups. the GISTIC2 peak region (where applicable) is listed in brackets after the c, Significant regions of focal SCNA identified by GISTIC2 in either pan-cohort suspected driver gene, as is the frequency of each event. or subgroup-specific analyses. d, e, Recurrent high-level amplifications a MYCL1 [6] SHH bc1106 LMO4 [9] e P P MB-1040 MB-698 MB-643 MB-391 MB-1249 MB-1133 MB-749 MB-7 MB-875 MB-486 MB-198 MB-1129 MB-1236 MB-488 MB-1276 MB-216 MB-539 MB-749 MB- MB-903 MB-1267 MB-488 MB-875 MB-347 MB-1094 MB-413 MB-1233 MB-184 MB-862 MB-1037 MB-698 MB-434 MB-1311 MB-14 MB-103 SHH signalling: 18.0% ( = 0.0009) RTK/PI3K signalling: 10.0% ( = 0.0139) Chr17Mb Chr1Mb 1q21.3[11] MDM4 [14] PTCH1 KIT 2p23.3[28] MYCN [2] PPP2R2B PDGFRA IGF1R GLI2 5.4% (7.7%) 0.4% (1.5%) 1.5% (4.9%) 1.9% (4.1%) 1.1% (4.9%) 4p16.3[126] [1] ACVR2B ELF2 [18] GLI1 GLI2 [3] PIK3C2B/MDM4 55.0–57.0 MYCN 3q25.1[8] 201.0–202.0 0.8% (0.8%) 5.3% (14%) 5q35.3[105] 8.3% (18%) IRS2 KIT [2] PPM1D 7p22.3[82] 1.9% (7.5%) PPP2R2B [1] 7q11.23[83] miR-17/92 CDKN2A/B 6p21.2[2] CCND2 8p12[3] 1.1% (7.5%) 1.5% (8.3%) 6q24.3[1] 3.4% (3.8%) 9p23[3] PIK3C2G CDK6 [7] CDKN2A/B P 1.1% (1.5%) [18] 8q22.1[2] TP53 signalling: 9.4% ( = 0.0012) PIK3C2B PTEN1 PTCH1 [5] YAP1 [1] CDKN2A/B 1.5% (7.5%) 1.5% (8.3%) KLF4 YAP1 [10] 12p13.33[35] 1.5% (8.3%) 1.5% (3.4%) 10p14[4] CCND2 [2] MDM1 MDM4 10p11.23[4] 12p12.1[4] AKT3 PTEN PPM1D PIK3C2B/MDM4 0.8% (0.8%) 1.5% (7.5%) [5] 12q15[1] 0.8% (6.8%) 15q15.1[24] IRS2 [3] TP53 TP53 IGF1R d 3.0% (26%) % of cases [4] [6] TSC1 >3.0 0 >3.0 17p11.2[26] 16p13.2[223] KLF4 PPM1D 1.1% (36%) Deleted Amplified 17p11.2[69] 1.1% (36%) 2.3%