Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

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We would like to thank all patients who have donated samples for this study. We also thank the team of the Biomedical Sequencing Facility at CeMM for support with next-generation sequencing; the members of the Delattre, Kovar, and Bock labs for discussions; A. Rendeiro and C. Dietz for contributing to the analysis pipelines; K. Clement for sharing his implementation of the PDR score; A. Lankester for providing MSCs; and the following physicians for providing tumor samples: J.M. Guinebretière, L. Brugières, A. de Muret, R. Tichit, N. Sirvent, F. Millot, F. Guilhot, J.P. Vannier, C. Michot, E. Plouvier, A. Gomez-Brouchet, J. Rivel, B. Petit, F. Dijoud, F. Larousserie, A. Kurt, A. Foulet, A.S. Desfachelles, H. Sartelet, I. Quintin Roue, J. Otten, J. Chasles, C. Bouvier, C. Soler, M. Peuchmaur, and X. Rialland. This study was funded by a grant from the Austrian National Bank's Jubiläumsfonds to E.M.T. (OeNB project number: 15714) and by a peer-reviewed institutional grant to E.M.T., which was based on a charitable donation of the Kapsch group (http://www.kapsch.net/kapschgroup) to St. Anna Kinderkrebsforschung. The French samples were collected in the context of the Plateforme Hospitalière de Génétique Moléculaire des Cancers of the Institut Curie and Centre Hospitalier de Versailles, with support by grants from INSERM within the framework of the International Cancer Genome Consortium program and from the Ligue Nationale Contre Le Cancer (Equipe labellisée), and the Société Française des Cancers de l'Enfant. The following associations supported this work: Courir pour Mathieu, Dans les pas du Géant, Olivier Chape, Les Bagouzamanon, Enfants et Santé, and les Amis de Claire. The study was performed in the context of the following European Union consortia: Euro Ewing (grant agreement no. 602856), BLUEPRINT (grant agreement no. 282510), PROVABES (grant agreement no. 01KT1310), ASSET (grant agreement no. 259348), and TECHNOBEAT (grant agreement no. 668724). N.C.S. was supported by a long-term fellowship of the Human Frontier Science Program (LT000211/2014). J.K. was supported by a DOC Fellowship of the Austrian Academy of Sciences. D. Surdez was supported by the Institut Curie-SIRIC (Site de Recherche Intégrée en Cancérologie) program. E.d.A. was supported by Ministry of Economy and Competitiveness of Spain-FEDER grants (CIBERONC, RD12/0036/0017, PI14/01466), María García-Estrada, CRIS contra el Cáncer Foundations, and Pablo Ugarte Association. C.B. was supported by a New Frontiers Group award of the Austrian Academy of Sciences and by a European Research Council (ERC) Starting Grant (European Union's Horizon 2020 research and innovation program; grant 679146). E.M.T. was supported by fellowships of the Austrian Science Fund (FWF, Lise Meitner Fellowship M1448-B13; and Elise Richter Fellowship V506-B28).

Author information

Author notes

    • Nathan C Sheffield

    Present address: Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, USA.

    • Olivier Delattre
    • , Heinrich Kovar
    • , Christoph Bock
    •  & Eleni M Tomazou

    These authors jointly directed this work.


  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Nathan C Sheffield
    • , Johanna Klughammer
    • , Paul Datlinger
    • , Andreas Schönegger
    • , Michael Schuster
    • , Johanna Hadler
    •  & Christoph Bock
  2. Institut Curie, PSL Research University, Service de Genetique, Pole de Medecine Diagnostique et Theranostique, Unité de Génétique Somatique, Paris, France.

    • Gaelle Pierron
    • , Delphine Guillemot
    • , Eve Lapouble
    • , Valérie Laurence
    •  & Olivier Delattre
  3. Institut Curie, PSL Research University, INSERM, U830, Paris, France.

    • Didier Surdez
    •  & Olivier Delattre
  4. Institut Curie, PSL Research University, Service de Pathologie, Pole de Medecine Diagnostique et Theranostique, Paris, France.

    • Paul Freneaux
  5. Service d'Anatomie et de Cytologie Pathologiques, Hopitaux de Brabois, Hopital d'Adultes, Nancy, France.

    • Jacqueline Champigneulle
  6. Centre de Pathologie du Pôle Est, Hopitaux de Lyon, Lyon, France.

    • Raymonde Bouvier
  7. Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.

    • Diana Walder
    • , Ingeborg M Ambros
    • , Eva Sorz
    • , Ruth Ladenstein
    • , Wolfgang Holter
    • , Peter F Ambros
    • , Heinrich Kovar
    •  & Eleni M Tomazou
  8. St. Anna Children's Hospital, St. Anna Kinderspital, Vienna, Austria.

    • Caroline Hutter
    • , Ruth Ladenstein
    •  & Wolfgang Holter
  9. Department of Pediatrics, Medical University of Vienna, Vienna, Austria.

    • Caroline Hutter
    • , Ruth Ladenstein
    • , Wolfgang Holter
    • , Peter F Ambros
    •  & Heinrich Kovar
  10. Department of Pathology, Institute of Biomedicine of Sevilla (IBiS), Virgen del Rocio University Hospital/CSIC/University of Sevilla, Seville, Spain.

    • Ana T Amaral
    •  & Enrique de Álava
  11. Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria.

    • Katharina Schallmoser
    •  & Dirk Strunk
  12. Department of Blood Group Serology and Transfusion Medicine, Paracelsus Medical University, Salzburg, Austria.

    • Katharina Schallmoser
  13. Institute for Experimental and Clinical Cell Therapy, Paracelsus Medical University, Salzburg, Austria.

    • Dirk Strunk
  14. Division of Biomedical Research, Medical University of Graz, Graz, Austria.

    • Beate Rinner
  15. Institute of Pathology, Medical University of Graz, Graz, Austria.

    • Bernadette Liegl-Atzwanger
  16. Organizational Unit of Research Infrastructure, Biobank Graz, Medical University of Graz, Graz, Austria.

    • Berthold Huppertz
  17. Department of Orthopedic Surgery, Medical University of Graz, Graz, Austria.

    • Andreas Leithner
  18. Service d'Anatomie et de Cytologie Pathologiques, Hôpital Universitaire Trousseau, Tours, France.

    • Gonzague de Pinieux
  19. Gustave Roussy, Département de Pathologie, Villejuif, France.

    • Philippe Terrier
  20. Institut Curie, PSL Research University, Departement d'Oncologie Pédiatrique Adolescent Jeunes Adultes, Paris, France.

    • Valérie Laurence
    •  & Jean Michon
  21. Department of Orthopedics, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.

    • Reinhard Windhager
  22. University Hospital Münster, Department of Pediatrics and Pediatric Hematology and Oncology, Münster, Germany.

    • Uta Dirksen
  23. Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.

    • Christoph Bock
  24. Max Planck Institute for Informatics, Saarland Informatics Campus, Saarbrücken, Germany.

    • Christoph Bock
  25. Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria.

    • Christoph Bock


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N.C.S., O.D., H.K., C.B., and E.M.T. designed the study. N.C.S. performed the data analysis with contributions from J.K., A.S., and M.S. G.P., D. Surdez, D.G., E.L., P.F., J.C., R.B., I.M.A., C.H., E.S., A.T.A., E.d.A., K.S., D. Strunk, B.R., B.L.-A., B.H., A.L., G.d.P., P.T., V.L., J.M., R.L., W.H., R.W., U.D., P.F.A., and O.D. provided materials such as tumor samples, clinical data, cell lines, and MSC samples. P.D., J.H., D.W., and E.M.T. performed the experiments. N.C.S., C.B., and E.M.T. wrote the manuscript with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Christoph Bock or Eleni M Tomazou.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12

Excel files

  1. 1.

    Supplementary Table 1

    Sample annotations and clinical data for the analyzed EwS tumors, EwS cell lines, and MSC samples.

  2. 2.

    Supplementary Table 2

    Data processing statistics for the RRBS experiments on EwS tumors, EwS cell lines, and MSC samples performed in this study (sheet 1); for publicly available RRBS profiles of various cancer samples (sheet 2); and for a diverse collection of cell types (sheet 3).

  3. 3.

    Supplementary Table 3

    Full results of the LOLA analyses (Figure 1d,e), showing enriched region sets among EwS hypomethylated CpGs (sheet 1) and among EwS hypermethylated CpGs (sheet 2), based on the LOLA Core database.

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