DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Abstract

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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Figure 1: DNA methylation profiling reveals a characteristic epigenomic signature of Ewing sarcoma.
Figure 2: DNA methylation in EwS shows inter-individual heterogeneity without distinct subtypes.
Figure 3: DNA methylation at regulatory elements defines an epigenetic disease spectrum underlying EwS.
Figure 4: DNA methylation patterns identify widespread intra-tumor heterogeneity in EwS.
Figure 5: DNA methylation heterogeneity in EwS is associated with genetic and clinical data.

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Acknowledgements

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).

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Authors

Contributions

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.

Corresponding authors

Correspondence to Christoph Bock or Eleni M Tomazou.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 13593 kb)

Supplementary Table 1

Sample annotations and clinical data for the analyzed EwS tumors, EwS cell lines, and MSC samples. (XLSX 27 kb)

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). (XLSX 101 kb)

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. (XLSX 802 kb)

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Sheffield, N., Pierron, G., Klughammer, J. et al. DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma. Nat Med 23, 386–395 (2017). https://doi.org/10.1038/nm.4273

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