Abstract
Recent sequencing efforts have described the mutational landscape of the pediatric brain tumor medulloblastoma. Although MLL2 is among the most frequent somatic single nucleotide variants (SNV), the clinical and biological significance of these mutations remains uncharacterized. Through targeted re-sequencing, we identified mutations of MLL2 in 8 % (14/175) of MBs, the majority of which were loss of function. Notably, we also report mutations affecting the MLL2-binding partner KDM6A, in 4 % (7/175) of tumors. While MLL2 mutations were independent of age, gender, histological subtype, M-stage or molecular subgroup, KDM6A mutations were most commonly identified in Group 4 MBs, and were mutually exclusive with MLL2 mutations. Immunohistochemical staining for H3K4me3 and H3K27me3, the chromatin effectors of MLL2 and KDM6A activity, respectively, demonstrated alterations of the histone code in 24 % (53/220) of MBs across all subgroups. Correlating these MLL2- and KDM6A-driven histone marks with prognosis, we identified populations of MB with improved (K4+/K27−) and dismal (K4−/K27−) outcomes, observed primarily within Group 3 and 4 MBs. Group 3 and 4 MBs demonstrate somatic copy number aberrations, and transcriptional profiles that converge on modifiers of H3K27-methylation (EZH2, KDM6A, KDM6B), leading to silencing of PRC2-target genes. As PRC2-mediated aberrant methylation of H3K27 has recently been targeted for therapy in other diseases, it represents an actionable target for a substantial percentage of medulloblastoma patients with aggressive forms of the disease.
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Acknowledgments
MDT is supported by a CIHR Clinician Scientist Phase II award. MDT and WW are supported by a grant from the National Institutes of Health (R01CA148699) and from The Pediatric Brain Tumor Foundation. Marc Remke is funded by the Mildred-Scheel Foundation/German Cancer Aid. We thank Susan Archer for assistance with technical writing.
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Supplementary material 4 Supplementary Fig. 1. Bioinformatic workflow used to discover and verify the presence of MLL2 and KDM6A mutations across 175 primary medulloblastomas. Mutations were inferred using SNVmix2 and SamTools INDEL detection metrics. Mutations, following filtering through SNP databases, were verified using Sanger sequencing. (EPS 1,312 kb)
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Supplementary material 5 Supplementary Fig. 2. Molecular characteristics of MLL2 expression, copy number and mutational analysis across multiple medulloblastomas cohorts. a, Subgroup-specific analysis of clonal and subclonal MLL2 mutations, based on sequence reads for wild-type and mutated alleles, reveals no homozygous MLL2 mutations. b, Sanger sequencing validation of representative MLL2 mutations, with corresponding read (wild-type versus mutated) counts. c, Frequency of MLL2 mutations per tumor genome across medulloblastoma discovery and validation cohorts demonstrates only SHH tumors have > 1 MLL2 mutation/genome. d, Expression analysis across two independent transcriptome cohorts indicates reduced MLL2 expression in WNT and SHH tumors and relatively higher expression in Group3 and Group 4 medulloblastomas. e, Copy number analysis of MLL2 genomic loci (chr12q13.12) across 201 primary medulloblastomas profiled on Affymterix 100 K and 500 K SNP arrays reveals no copy number alterations (i.e., deletions, 1.5 < MLL2 CN > 2.75). (EPS 4,778 kb)
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Supplementary material 6 Supplementary Fig. 3. Clinical characteristics of medulloblastomas with MLL2 mutations or MLL2 immunonegativity. a, Kaplan–Meier analysis of MLL2 mutations across 175 primary medulloblastomas reveals no significant (P < 0.80) difference in overall survival associated with MLL2 mutations. b, Kaplan–Meier survival analysis of MLL2 immunohistochemical staining across 239 medulloblastomas reveals no significant (P < 0.41) subgroup-independent survival differences between positively and negatively staining samples. (EPS 1,121 kb)
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Supplementary material 7 Supplementary Fig. 4. KDM6A mutational and expression characteristics across medulloblastomas cohorts. a, Subgroup-specific analysis of clonal and subclonal KDM6A mutations, based on sequence reads for wild-type and mutated alleles, reveals exclusively homozygous deletions in Group 4 medulloblastomas while SHH and Group 3 tumors demonstrate subclonal and heterozygous read frequencies. b, KDM6A gene expression profiling analysis across Toronto (n = 103) and Heidelberg (n = 302) medulloblastoma datasets reveals similar trends in expression with SHH tumors demonstrating the highest expression patterns. (EPS 2,467 kb)
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Supplementary material 8 Supplementary Fig. 5. Immunostaining for MLL2- and KDM6A-driven histone marks, H3K4me3 and H3K27me3 across medulloblastoma tissue microarrays. a, H3K4me3 staining patterns across Johns Hopkins and DKFZ tissue microarrays reveals no significant association between H3K4me3 positivity versus negativity with overall survival. Both arrays demonstrate trends towards decreased survival associated with H3K4me3-negativity. b, H3K27me3 immunostaining across Johns Hopkins and DKFZ tissue microarrays reveals no significant association between H3K27mme3 positivity versus negativity with overall survival. (EPS 9,357 kb)
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Supplementary material 9 Supplementary Fig. 6. Copy number and expression analysis of cytogenetic aberrations converging on H3K27me-modifiers across the medulloblastoma genome. a, Significant correlation between copy number and expression of EZH2 in both a subgroup-independent and dependent analyses. b, While KDM6A and KDM6B demonstrates trends towards chrX loss (KDM6A) or chr17p loss (KDM6B) and reduced expression in a subgroup dependent manner. (EPS 3,063 kb)
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Dubuc, A.M., Remke, M., Korshunov, A. et al. Aberrant patterns of H3K4 and H3K27 histone lysine methylation occur across subgroups in medulloblastoma. Acta Neuropathol 125, 373–384 (2013). https://doi.org/10.1007/s00401-012-1070-9
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DOI: https://doi.org/10.1007/s00401-012-1070-9