Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma

Abstract

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

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Figure 1: Genomic landscape of pediatric midline HGAs.
Figure 2: Increased levels of phosphorylated SMAD1/5/8 in ACVR1-mutant mHGAs.
Figure 4: Mutations identified in ACVR1 are associated with activation of downstream SMAD signaling pathways.
Figure 3: Clustering analysis of global DNA methylation profiles for 98 high-grade astrocytomas.

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Acknowledgements

The authors would like to express their sincere gratitude toward all staff at the McGill University and Génome Québec Innovation Centre for excellent technical expertise, library preparation and sequencing. The authors are very grateful to J.-J. Lebrun (McGill University) for primer sequences and materials for SMAD signaling studies. This work was performed within the context of the I-CHANGE (International Childhood Astrocytoma Integrated Genomics and Epigenomics) Consortium and was supported by funding from Genome Canada, Génome Québec, the Institute for Cancer Research of the Canadian Institutes for Health Research (CIHR), McGill University and the Montreal Children's Hospital Foundation. This work was also supported by Hungarian Scientific Research Fund (OTKA) contract T-04639, National Research and Development Fund (NKFP) contract 1A/002/2004 (P.H. and M.G.) and TÁMOP-4.2.2A-11/1/KONV-2012-0025 (A.K. and L.B.). N.J. is a member of the Penny Cole laboratory and the recipient of a Chercheur Clinicien Senior Award. J. Majewski holds a Canada Research Chair (tier 2). L.G., K.L.L. and M.W.K. are supported by NCI P01CA142536. We acknowledge the support of the Zach Carson DIPG Fund at the Dana-Farber Cancer Institute (DFCI), the Ellie Kavalieros Fund (DFCI), the Mikey Czech Foundation, the Prayer From Maria Foundation, the Hope for Caroline Fund (DFCI), the Ryan Harvey DIPG Fund (DFCI), the Stop&Shop Pediatric Brain Tumor Program (DFCI) and the Pediatric Brain Tumor Clinical and Research Fund (DFCI). A.M.F. is supported by a studentship from CIHR, as well as by an award from the CIHR Systems Biology Training Program at McGill University. D.B. is supported by a studentship from the T.D. Trust/Montreal Children's Hospital Foundation, and N. Gerges is supported by a studentship from the Cedars Cancer Institute. N.D.J. is supported by an award from the McGill Integrated Cancer Research Training Program.

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A.M.F., N. Gerges, P.-O.F., D.B., D.F., L.A.R., A.C., A.H.L., S.A. and Z.D. performed experiments. A.M.F., S.P.-C., J.S., H.N., N.D.J., A.H.L., S.A., Z.D. and P.M.S. analyzed the data and produced figures and tables. D.T.W.J., D.S., P.J., T.T., S.G., M.N., A.B., L.G., D.C.B., J.R.L., J.B.R., T.A., S.B., J.R.G., G.J., K.C., N. Gupta, M.D.P., A.-S.C., B.E., L.C., A.K., L.B., P.H., M.G., J. Myseros, H.M., S.A. and S.M.P. provided tissue samples. K.L.L., J. Majewski, N.J. and M.W.K. provided project leadership and designed the study. All authors contributed to the final manuscript.

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Correspondence to Keith L Ligon or Jacek Majewski or Nada Jabado or Mark W Kieran.

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Integrated supplementary information

Supplementary Figure 1 Patterns of K27M-associated mutations among midline pediatric high-grade astrocytomas.

a, Neuroanatomical distribution of mutations and alterations of interest in pediatric midline high-grade astrocytomas. b, The age of patients harboring H3.3 and H3.1 mutations is significantly different (P = 0.0280; two-tailed t test). c, Mutual exclusivity and associative statistical analyses (represented as P values) of mutations in H3F3A, HIST1H3B/HIST1H3C, TP53, FGFR1, ACVR1, PDGFRA and PI3K pathway genes PIK3CA/PIK3R1/PTEN demonstrates predilection for co-occurrence of and exclusion of particular mutations as unique mutational groups within 39 midline high-grade astrocytomas profiled by whole-exome sequencing. P values were calculated utilizing Fisher's exact test (two-sided) with highlighted values indicating statistically significant (P < 0.05) preferences for co-occurrence or mutual exclusivity.

Supplementary Figure 2 PDGFRA amplification is present in a minority of cells in DIPG.

Representative images from FISH assay for PDGFRA amplification reveal substantial increase in the number of PDFRA-positive nuclei following treatment compared with samples from pretreatment biopsies. Green, PDGFRA; red, Chr4/CEN4.

Supplementary Figure 3 Copy number variant analysis using biopsy material from five different anatomical loci in a single tumor.

a, Tumor biopsies (n = 5) show highly similar CNV patterns across the genome. b, Plots depicting 20-Mb zoom-in of chromosome 4, showing significant PDGFRA, KIT and KDR amplification in only the deep tissue biopsy and not others.

Supplementary Figure 4 H3.1 and H3.3 K27M variants demonstrate similar patterns of global DNA methylation.

Heat map of hierarchical clustering analysis of DNA methylation array data from the top 10,000 most variable β values from 27 K27M-mutant high-grade gliomas demonstrates that H3.3 and H3.1 K27M mutant tumors show similar patterns of global epigenomic dysregulation.

Supplementary Figure 5 Multiscale bootstrapping of the high-grade astrocytoma methylation cluster.

Dendrogram of multiscale bootstrapping of DNA methylation profiles of patient high-grade tumors. P values, represented as the red values at the internal nodes, are the approximately unbiased (AU) P value computed by the R package pvclust.

Supplementary Figure 6 Mutation subgroup-specific overall survival of midline high-grade astrocytomas (mHGAs) and diffuse intrinsic pontine gliomas (DIPGs).

Kaplan-Meier analysis of overall survival (months) of mHGAs with available outcome data, with indicated P values from Mantel-Cox (log-rank) and Gehan-Breslow-Wilcoxon testing for comparison based on a, ACVR1 mutation status (n = 27), b, H3.3/H3.1 K27M mutation status (n = 27) and overall survival restricted to tumors of the pontine area based on c, ACVR1 mutation status (n = 23), d, H3.3/H3.1 K27M mutation status (n = 22).

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Fontebasso, A., Papillon-Cavanagh, S., Schwartzentruber, J. et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat Genet 46, 462–466 (2014). https://doi.org/10.1038/ng.2950

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