Abstract

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers.

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Acknowledgements

We thank the Children's Oncology Group for the collection and annotation of samples for this study, and all TARGET co-investigators for scientific support of this project. Funding was provided by US National Institutes of Health grants CA98543 and CA98413 to the Children's Oncology Group, RC1MD004418 to the TARGET consortium, CA124709 (J.M.M.) and CA060104 (R.C.S.) and National Human Genome Research Institute grant U54HG003067 (E.S.L., D.A., S.B.G., G.G. and M.M.), as well as a contract from the National Cancer Institute, US National Institutes of Health (HHSN261200800001E). Additional support included a Canadian Institutes of Health Research Fellowship (T.J.P.), a Roman M. Babicki Fellowship in Medical Research at the University of British Columbia (O.M.), the Canada Research Chair in Genome Science (M.A.M.), the Giulio D'Angio Endowed Chair (J.M.M.), the Alex's Lemonade Stand Foundation (J.M.M.), the Arms Wide Open Foundation (J.M.M.) and the Cookies for Kids Foundation (J.M.M.). We thank E. Nickerson, S. Channer, K. Novik, C. Suragh and R. Roscoe for project management support. We also thank the staff of the Genome Sciences Centre Biospecimen Core, Library Construction, Sequencing and Bioinformatics teams, and the staff of the Broad Institute Biological Samples, Genome Sequencing and Genetic Analysis Platforms for their expertise in genomic processing of samples, and generating the sequencing data used in this analysis.

Author information

Author notes

    • Trevor J Pugh
    •  & Olena Morozova

    These authors contributed equally to this work.

Affiliations

  1. The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Trevor J Pugh
    • , Daniel Auclair
    • , Scott L Carter
    • , Kristian Cibulskis
    • , Megan Hanna
    • , Adam Kiezun
    • , Jaegil Kim
    • , Michael S Lawrence
    • , Lee Lichenstein
    • , Aaron McKenna
    • , Chandra Sekhar Pedamallu
    • , Alex H Ramos
    • , Erica Shefler
    • , Andrey Sivachenko
    • , Carrie Sougnez
    • , Chip Stewart
    • , Eric S Lander
    • , Stacey B Gabriel
    • , Gad Getz
    •  & Matthew Meyerson
  2. Harvard Medical School, Boston, Massachusetts, USA.

    • Trevor J Pugh
    • , Alex H Ramos
    • , Wendy B London
    •  & Matthew Meyerson
  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Trevor J Pugh
    • , Megan Hanna
    • , Chandra Sekhar Pedamallu
    •  & Matthew Meyerson
  4. Genome Sciences Centre, British Columbia Cancer Agency, University of British Columbia, Vancouver, British Columbia, Canada.

    • Olena Morozova
    • , Adrian Ally
    • , Inanc Birol
    • , Readman Chiu
    • , Richard D Corbett
    • , Martin Hirst
    • , Shaun D Jackman
    • , Baljit Kamoh
    • , Alireza Hadj Khodabakshi
    • , Martin Krzywinski
    • , Allan Lo
    • , Richard A Moore
    • , Karen L Mungall
    • , Jenny Qian
    • , Angela Tam
    • , Nina Thiessen
    • , Yongjun Zhao
    • , Steven J M Jones
    •  & Marco A Marra
  5. Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

    • Olena Morozova
    •  & Marco A Marra
  6. Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

    • Edward F Attiyeh
    • , Kristina A Cole
    • , Maura Diamond
    • , Sharon J Diskin
    • , Yael P Mosse
    • , Andrew C Wood
    • , Michael D Hogarty
    •  & John M Maris
  7. Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

    • Edward F Attiyeh
    • , Kristina A Cole
    • , Maura Diamond
    • , Sharon J Diskin
    • , Yael P Mosse
    • , Andrew C Wood
    • , Michael D Hogarty
    •  & John M Maris
  8. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Edward F Attiyeh
    • , Kristina A Cole
    • , Maura Diamond
    • , Sharon J Diskin
    • , Yael P Mosse
    • , Andrew C Wood
    • , Michael D Hogarty
    •  & John M Maris
  9. Division of Hematology/Oncology, The Children's Hospital Los Angeles, Los Angeles, California, USA.

    • Shahab Asgharzadeh
    • , Lingyun Ji
    • , Richard Sposto
    •  & Robert C Seeger
  10. Saban Research Institute, The Children's Hospital Los Angeles, Los Angeles, California, USA.

    • Shahab Asgharzadeh
    • , Lingyun Ji
    • , Richard Sposto
    •  & Robert C Seeger
  11. Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Shahab Asgharzadeh
    • , Lingyun Ji
    • , Richard Sposto
    •  & Robert C Seeger
  12. Pediatric Oncology Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Gaithersburg, Maryland, USA.

    • Jun S Wei
    • , Thomas Badgett
    •  & Javed Khan
  13. Children's Hospital Boston/Dana-Farber Cancer Institute and Children's Oncology Group, Boston, Massachusetts, USA.

    • Wendy B London
  14. Biopathology Center, Nationwide Children's Hospital, Columbus, Ohio, USA.

    • Yvonne Moyer
    •  & Julie M Gastier-Foster
  15. The Ohio State University College of Medicine, Columbus, Ohio, USA.

    • Yvonne Moyer
    •  & Julie M Gastier-Foster
  16. Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, Maryland, USA.

    • Malcolm A Smith
  17. Office of Cancer Genomics, National Cancer Institute, Bethesda, Maryland, USA.

    • Jaime M Guidry Auvil
    •  & Daniela S Gerhard
  18. Abramson Family Cancer Research Institute, Philadelphia, Pennsylvania, USA.

    • John M Maris

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Contributions

J.M.M., J. Khan, R.C.S., D.S.G. and M.A.S. conceived and led the project. M.A.M. and M.M. conceived of and supervised all aspects of the sequencing work at the British Columbia (BC) Cancer Agency Genome Sciences Centre and Broad Institute, respectively. T.J.P. and O.M. performed the analyses and interpreted the results. E.F.A., S.A., J.S.W., K.A.C., M.D., S.J.D., A.C.W., Y.P.M., L.J., T.B., Y.M., J.M.G.-F. and M.D.H. selected and characterized samples, provided disease-specific expertise in data analysis and edited the manuscript. R.S. and W.B.L. provided statistical support and analyses of clinical covariates. D.A., E.S., C. Sougnez, M.D. and J.M.G.A. provided overall project management and quality control support. S.L.C., K.C., M. Hanna, A.K., J. Kim, M.S.L., L.L., A.M., A.H.R., A.S. and C. Stewart supported analysis of somatic and germline alterations in the exome sequencing data. C.S.P. performed the pathogen discovery analysis. I.B., K.L.M., R.C., S.D.J. and J.Q. performed de novo assembly of Illumina sequencing data. Y.Z. led the library construction effort for the Illumina libraries. A.T. and Y.Z. planned the sequencing verification, and A.A. and B.K. performed the experiments. R.D.C. performed copy number analysis of genome sequencing data. M.K. performed verification of candidate rearrangements. N.T. performed gene- and exon-level quantification analysis of RNA-seq data. A.L. and A.H.K. helped interpret data provided by Complete Genomics. R.A.M. and M. Hirst led the sequencing effort for the Illumina genome and transcriptome libraries. S.B.G. and E.S.L. led the sequencing effort for the exome sequencing libraries. G.G. and S.J.M.J. supervised the bioinformatics group at the Broad Institute and BC Cancer Agency Genome Sciences Centre, respectively. T.J.P., O.M., D.S.G., M.A.M., M.M. and J.M.M. cowrote the manuscript with input from all coauthors.

Competing interests

M.M. is a paid consultant for and equity holder in Foundation Medicine, a genomics-based oncology diagnostics company, and is a paid consultant for Novartis.

Corresponding authors

Correspondence to Marco A Marra or Matthew Meyerson or John M Maris.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Note, Supplementary Tables 11–14 and Supplementary Figures 1–10

Excel files

  1. 1.

    Supplementary Table 1

    Master data table: Clinical and molecular data for all neuroblastoma cases including identifiers from other databases, sequencing technologies used, clinical and biological covariates, and matrix of mutation calls

  2. 2.

    Supplementary Table 2

    Coverage: Fraction of bases in each exon with sufficient coverage for mutation detection

  3. 3.

    Supplementary Table 3

    Full mutation list: All coding somatic mutations called in all cases

  4. 4.

    Supplementary Table 4

    Mutation frequency correlates: Statistical comparison of mutation frequency distributions (Kolmogorov-Smirnov) when comparing cases by clinical and biological variables

  5. 5.

    Supplementary Table 5

    Pathogens: Counts of sequencing reads in exome capture libraries corresponding to known viruses

  6. 6.

    Supplementary Table 6

    MutSig: Significance analysis of somatic mutation frequency in all genes and a focused set of genes listed in the Catalogue of Somatic Mutations in Cancer

  7. 7.

    Supplementary Table 7

    Gene set significance analysis: Full list of pathways, member genes, mutated genes, and significance values as calculated by MutSig with and without significantly mutated genes

  8. 8.

    Supplementary Table 8

    Structural rearrangements: All structural variants detected in neuroblastoma genomes or transcriptomes

  9. 9.

    Supplementary Table 9

    Significance analysis of germline ClinVar variation: List of all genes tested for enrichment in neuroblastoma of ClinVar variants

  10. 10.

    Supplementary Table 10

    Significance analysis of germline loss-of-function variants in Cancer Census, cancer syndrome, or DNA repair genes

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DOI

https://doi.org/10.1038/ng.2529

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