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The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space

Nature Medicine volume 24, pages 1611–1624 (2018)Cite this article

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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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Fig. 1: DNA methylation landscape of glioblastoma disease progression.
Fig. 2: Glioblastoma transcriptional subtypes inferred from DNA methylation profiles.
Fig. 3: DNA methylation and the tumor microenvironment.
Fig. 4: DNA methylation and histopathological tumor characteristics.
Fig. 5: DNA methylation heterogeneity in glioblastoma disease progression.
Fig. 6: DNA methylation differences between primary and recurring tumors.

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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.

Authors and 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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  1. Johanna Klughammer
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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.

Corresponding author

Correspondence to Adelheid Woehrer.

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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.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13

Reporting Summary

Supplementary Table 1

Patient summary table

Supplementary Table 2

RRBS summary table

Supplementary Table 3

Survival analysis summary table

Supplementary Table 4

Association analysis summary table

Source Data Figure 1

Source Data Figure 1

Source Data Figure 2

Source Data Figure 2

Source Data Figure 3

Source Data Figure 3

Source Data Figure 4

Source Data Figure 4

Source Data Figure 5

Source Data Figure 5

Source Data Figure 6

Source Data Figure 6

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Klughammer, J., Kiesel, B., Roetzer, T. et al. The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space. Nat Med 24, 1611–1624 (2018). https://doi.org/10.1038/s41591-018-0156-x

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

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