Abstract

Glioblastoma is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the role of the epigenome in glioblastoma disease progression. Here, we present genome-scale maps of DNA methylation in matched primary and recurring glioblastoma tumors, using data from a highly annotated clinical cohort that was selected through a national patient registry. We demonstrate the feasibility of DNA methylation mapping in a large set of routinely collected FFPE samples, and we validate bisulfite sequencing as a multipurpose assay that allowed us to infer a range of different genetic, epigenetic, and transcriptional characteristics of the profiled tumor samples. On the basis of these data, we identified subtle differences between primary and recurring tumors, links between DNA methylation and the tumor microenvironment, and an association of epigenetic tumor heterogeneity with patient survival. In summary, this study establishes an open resource for dissecting DNA methylation heterogeneity in a genetically diverse and heterogeneous cancer, and it demonstrates the feasibility of integrating epigenomics, radiology, and digital pathology for a national cohort, thereby leveraging existing samples and data collected as part of routine clinical practice.

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Acknowledgements

We thank all patients who have donated their samples for this study. We also thank G. Wilk, M. Muck, S. Schmid, and U. Andel for technical assistance with immunohistochemical stainings, macrodissection, and tumor tissue shavings; S. Mages for contributing to the interactive data visualization; the Biomedical Sequencing Facility at CeMM for assistance with next-generation sequencing; and all members of the Bock lab for their help and advice. The study was funded in part by an Austrian Science Fund grant (FWF KLI394) to A.W., a Marie Curie Career Integration Grant (European Union’s Seventh Framework Programme grant agreement no. PCIG12-GA-2012-333595) to C.B., an ERA-NET project grant (EpiMark FWF I 1575-B19) to C.B., an Austrian Science Fund grant (FWF I2714-B31) to G.L. and K.-H.N, and an ERC Starting Grant (European Union’s Horizon 2020 research and innovation programme, grant agreement no. 640396) to B.B. Moreover, C.B. is supported by a New Frontiers Group award of the Austrian Academy of Sciences and by an ERC Starting Grant (European Union’s Horizon 2020 research and innovation programme, grant agreement no. 679146). Activities of the Austrian Brain Tumor Registry are supported by unrestricted research grants of Roche Austria to J.A.H. and the Austrian Society of Neurology to S.O. Some of the samples used for this research project were kindly provided by Biobank Graz.

Author information

Author notes

  1. These authors contributed equally to this work: Johanna Klughammer, Barbara Kiesel.

  2. These authors jointly supervised this work: Adelheid Woehrer, Christoph Bock.

Affiliations

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

    • Johanna Klughammer
    • , Nikolaus Fortelny
    • , Amelie Nemc
    • , Paul Datlinger
    • , Donat Alpar
    • , Bekir Ergüner
    • , Martin Senekowitsch
    •  & Christoph Bock
  2. Department of Neurosurgery, Medical University of Vienna, Vienna, Austria

    • Barbara Kiesel
    • , Mario Mischkulnig
    • , Engelbert Knosp
    •  & Georg Widhalm
  3. Comprehensive Cancer Center, Central Nervous System Tumor Unit, Medical University of Vienna, Vienna, Austria

    • Barbara Kiesel
    • , Thomas Roetzer
    • , Julia Furtner
    • , Nadine Peter
    • , Mario Mischkulnig
    • , Thomas Ströbel
    • , Karin Dieckmann
    • , Matthias Preusser
    • , Engelbert Knosp
    • , Georg Widhalm
    • , Christine Marosi
    • , Johannes A. Hainfellner
    •  & Adelheid Woehrer
  4. Institute of Neurology, Medical University of Vienna, Vienna, Austria

    • Thomas Roetzer
    • , Nadine Peter
    • , Thomas Ströbel
    • , Johannes A. Hainfellner
    •  & Adelheid Woehrer
  5. Department of Biomedical Imaging and Image-guided Therapy, Computational Imaging Research Lab, Medical University of Vienna, Vienna, Austria

    • Karl-Heinz Nenning
    •  & Georg Langs
  6. Department of Biomedical Imaging and Image-guided Therapy, Division of Neuroradiology and Musculoskeletal Radiology, Medical University of Vienna, Vienna, Austria

    • Julia Furtner
  7. Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA

    • Nathan C. Sheffield
  8. Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria

    • Martha Nowosielski
    •  & Günther Stockhammer
  9. University Medical Center, Neurology, German Cancer Research Center, Heidelberg, Germany

    • Martha Nowosielski
  10. Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria

    • Marco Augustin
    •  & Bernhard Baumann
  11. Department of Pathology, Medical University of Innsbruck, Innsbruck, Austria

    • Patrizia Moser
    •  & Johannes Haybaeck
  12. Department of Neurosurgery, Medical University of Innsbruck, Innsbruck, Austria

    • Christian F. Freyschlag
    • , Johannes Kerschbaumer
    •  & Claudius Thomé
  13. Department of Neuroradiology, Medical University of Innsbruck, Innsbruck, Austria

    • Astrid E. Grams
  14. Department of Pathology, University Hospital of St. Poelten, Karl Landsteiner University of Health Sciences, St. Poelten, Austria

    • Melitta Kitzwoegerer
  15. Department of Neurology, University Hospital of St. Poelten, Karl Landsteiner University of Health Sciences, St. Poelten, Austria

    • Stefan Oberndorfer
  16. Department of Neurosurgery, University Hospital of St. Poelten, Karl Landsteiner University of Health Sciences, St. Poelten, Austria

    • Franz Marhold
  17. Department of Neuropathology, Neuromed Campus Wagner-Jauregg, Kepler University Hospital, Johannes Kepler University of Linz, Linz, Austria

    • Serge Weis
  18. Department of Neuroradiology, Neuromed Campus Wagner-Jauregg, Kepler University Hospital, Johannes Kepler University of Linz, Linz, Austria

    • Johannes Trenkler
  19. Department of Neurosurgery, Neuromed Campus Wagner-Jauregg, Kepler University Hospital, Johannes Kepler University of Linz, Linz, Austria

    • Johanna Buchroithner
  20. Department of Internal Medicine, Neuromed Campus Wagner-Jauregg, Kepler University Hospital, Johannes Kepler University of Linz, Linz, Austria

    • Josef Pichler
  21. Diagnostic & Research Center for Molecular BioMedicine, Department of Neuropathology, Institute of Pathology, Medical University of Graz, Graz, Austria

    • Johannes Haybaeck
    •  & Stefanie Krassnig
  22. Department of Pathology, Medical Faculty, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany

    • Johannes Haybaeck
  23. Department of Neurosurgery, Medical University of Graz, Graz, Austria

    • Kariem Mahdy Ali
    •  & Gord von Campe
  24. Department of Neurology, Medical University of Graz, Graz, Austria

    • Franz Payer
  25. Department of Neurosurgery, Krankenanstalt Rudolfstiftung, Vienna, Austria

    • Camillo Sherif
  26. Department of Pathology, Krankenanstalt Rudolfstiftung, Vienna, Austria

    • Julius Preiser
  27. Department of Neurosurgery, Christian-Doppler-Klinik, Paracelsus Private Medical University, Salzburg, Austria

    • Thomas Hauser
    •  & Peter A. Winkler
  28. Department of Neurology, Christian-Doppler-Klinik, Paracelsus Private Medical University, Salzburg, Austria

    • Waltraud Kleindienst
  29. Institute of Pathology, State Hospital Klagenfurt, Klagenfurt, Austria

    • Franz Würtz
    •  & Tanisa Brandner-Kokalj
  30. Department of Neurology, State Hospital Klagenfurt, Klagenfurt, Austria

    • Martin Stultschnig
  31. Department of Neurosurgery, General Hospital Wiener Neustadt, Wiener Neustadt, Austria

    • Stefan Schweiger
  32. Department of Radiotherapy, Medical University of Vienna, Vienna, Austria

    • Karin Dieckmann
  33. Department of Medicine I, Medical University of Vienna, Vienna, Austria

    • Matthias Preusser
    •  & Christine Marosi
  34. Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

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

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

    • Christoph Bock

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Contributions

J. Klughammer, A.W., and C.B. designed the study. B.K., T.R., K.-H.N., J.F., N.P., M.N., M.A., M.M., T.S., G.L., B.B., J.A.H., and A.W. established and annotated the cohort. A.N. and P.D. performed DNA methylation profiling. D.A. performed low-coverage whole-genome sequencing. M.S. performed RNA-seq. J. Klughammer performed the data analysis. N.F., N.C.S, and B.E. contributed to data analysis. P.M., C.F.F., J. Kerschbaumer, C.T., A.E.G., G.S., M.K., S.O., F.M., S.W., J.T., J.B., J. Pichler, J.H., S.K., K.M.A., G.v.C., F.P., C.S., J. Preiser, T.H., P.A.W., W.K., F.W., T.B.-K., M.S., S.S., K.D., M.P., E.K., G.W., and C.M. contributed tumor samples and clinical data. J. Klughammer, A.W., and C.B. wrote the manuscript with contributions from all authors.

Competing interests

The optimized RRBS protocol that was used in this study has been licensed to Diagenode s.a. (Liège, Belgium) and commercialized as a kit and service.

Corresponding author

Correspondence to Adelheid Woehrer.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–13

  2. Reporting Summary

  3. Supplementary Table 1

    Patient summary table

  4. Supplementary Table 2

    RRBS summary table

  5. Supplementary Table 3

    Survival analysis summary table

  6. Supplementary Table 4

    Association analysis summary table

  7. Source Data Figure 1

    Source Data Figure 1

  8. Source Data Figure 2

    Source Data Figure 2

  9. Source Data Figure 3

    Source Data Figure 3

  10. Source Data Figure 4

    Source Data Figure 4

  11. Source Data Figure 5

    Source Data Figure 5

  12. Source Data Figure 6

    Source Data Figure 6

About this article

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DOI

https://doi.org/10.1038/s41591-018-0156-x