Abstract

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n = 66) and high-grade dysplasia (HGD; n = 43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett's esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.

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Acknowledgements

Whole-genome sequencing of EAC is part of the International Cancer Genome Consortium (ICGC) through the Esophageal Cancer Clinical and Molecular Stratification (OCCAMS) Consortium and is funded by Cancer Research UK. We thank the ICGC members for their input on verification standards as part of the benchmarking exercise. Cytosponge samples were collected as part of the Cancer Research UK–funded BEST2 trial. We thank M. Griffin, L. Lovat and K. Ragunath for their contribution to Cytosponge collection. The MRC developed the Cytosponge and also funded laboratory work through a program grant to R.C.F. J.M.J.W. was funded by a Wellcome Trust Translational Medicine and Therapeutics grant. R.C.F. and C.C. are supported by additional clinical research infrastructure funding from the NHS National Institute for Health Research (NIHR), the Experimental Cancer Medicine Centre Network and the NIHR Cambridge Biomedical Research Centre. Bioinformatics work was also supported by a Cancer Research UK program grant to S.T.

We thank the Genomics Core at the Cancer Research UK Cambridge Institute for their help with processing some of the Access Array experiments as well as for running the targeted resequencing experiments. We thank the IT department at the Cancer Research UK Cambridge Institute for their support. We thank F. Marass for assistance with data analysis. We thank the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre, from Addenbrooke's Hospital as well as the University Hospital of Southampton Trust and the Southampton Experimental Cancer Medicine Centre. We are grateful to all patients who provided written consent for participation in this study, and the staff at Addenbrooke's and the University of Southampton Tissue Bank.

Author information

Author notes

    • Jamie M J Weaver
    • , Caryn S Ross-Innes
    • , Nicholas Shannon
    •  & Andy G Lynch

    These authors contributed equally to this work.

Affiliations

  1. Medical Research Council (MRC) Cancer Unit, University of Cambridge, Cambridge, UK.

    • Jamie M J Weaver
    • , Caryn S Ross-Innes
    • , Mariagnese Barbera
    • , Chin-Ann J Ong
    • , Pierre Lao-Sirieix
    • , Laura Smith
    • , Nicola Grehan
    • , Rebecca C Fitzgerald
    • , Olga Knight
    • , Barbara Nutzinger
    • , Zarah Abdullahi
    • , Irene Debriram-Beecham
    • , Jason Crawte
    • , Shona MacRae
    • , Ayesha Noorani
    • , Rachael Fels Elliott
    • , Xiaodun Li
    • , Jan Bornschein
    • , Sebastian Zeki
    • , Hamza Chettouh
    • , Nadeera de Silva
    • , Eleanor Gregson
    •  & Tsun-Po Yang
  2. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.

    • Nicholas Shannon
    • , Andy G Lynch
    • , Tim Forshew
    • , Muhammed Murtaza
    • , Mark J Dunning
    • , Mike L Smith
    • , Charlotte L Anderson
    • , Benilton Carvalho
    • , Carlos Caldas
    • , Matthew D Eldridge
    • , Nitzan Rosenfeld
    • , Simon Tavaré
    • , Lawrence Bower
    • , Achilleas Achilleos
    •  & Maria Secrier
  3. Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK.

    • Maria O'Donovan
    • , Alison Cluroe
    • , Ahmad Miremadi
    • , Betania Mahler-Araujo
    •  & Shalini Malhotra
  4. Cancer Sciences Division, University of Southampton, Southampton, UK.

    • Timothy J Underwood
  5. Fluidigm Corporation, South San Francisco, California, USA.

    • Andrew P May
  6. Oesophago-Gastric Unit, Addenbrooke's Hospital, Cambridge, UK.

    • Richard Hardwick
  7. Oxford Computing Laboratory, University of Oxford, Oxford, UK.

    • Jim Davies
  8. British Columbia Cancer Research Centre, Cancer Agency Research Centre, Vancouver, British Columbia, Canada.

    • Arusha Oloumi
    •  & Sam Aparicio
  9. Department of Pathology, University of Cambridge, Cambridge, UK.

    • Paul A W Edwards
  10. Salford Royal National Health Service (NHS) Foundation Trust, Salford, UK.

    • Stephen J Hayes
    • , Ang Yeng
    • , Anne-Marie Lydon
    •  & Soney Dharmaprasad
  11. Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.

    • Stephen J Hayes
  12. Wigan and Leigh NHS Foundation Trust, Manchester, UK.

    • Sandra Greer
  13. Royal Surrey County Hospital NHS Foundation Trust, Guildford, UK.

    • Shaun Preston
    •  & Sarah Oakes
  14. Edinburgh Royal Infirmary, Edinburgh, UK.

    • Vicki Save
    •  & Simon Paterson-Brown
  15. University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK.

    • Olga Tucker
    • , Derek Alderson
    •  & Philippe Taniere
  16. Department of Computer Science, University of Oxford, Oxford, UK.

    • Olga Tucker
    •  & Charles Crichton
  17. Southampton General Hospital, Southampton, UK.

    • Jamie Kelly
    • , James Byrne
    • , Donna Sharland
    • , Nina Holling
    • , Lisa Boulter
    • , Fergus Noble
    •  & Bernard Stacey
  18. Gloucester Royal Hospital, Gloucester, UK.

    • Hugh Barr
    • , Neil Shepherd
    • , L Max Almond
    •  & Oliver Old
  19. St Thomas's Hospital, London, UK.

    • Jesper Lagergren
    • , James Gossage
    • , Andrew Davies
    • , Robert Mason
    • , Fuju Chang
    • , Janine Zylstra
    •  & Chris Peters
  20. King's College London, London, UK.

    • Jesper Lagergren
    • , James Gossage
    • , Andrew Davies
    • , Robert Mason
    • , Fuju Chang
    •  & Janine Zylstra
  21. Karolinska Institutet, Stockholm, Sweden.

    • Jesper Lagergren
    • , James Gossage
    • , Andrew Davies
    •  & Robert Mason
  22. Plymouth Hospitals NHS Trust, Plymouth, UK.

    • Grant Sanders
    • , Tim Wheatley
    • , Richard Berrisford
    • , Tim Bracey
    • , Catherine Harden
    •  & David Bunting
  23. Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK.

    • Tom Roques
    • , Jenny Nobes
    • , Suat Loo
    • , Mike Lewis
    • , Ed Cheong
    •  & Oliver Priest
  24. Nottingham University Hospitals NHS Trust, Nottingham, UK.

    • Simon L Parsons
    • , Irshad Soomro
    • , Philip Kaye
    • , John Saunders
    • , Vincent Pang
    • , Neil T Welch
    • , James A Catton
    • , John P Duffy
    •  & Krish Ragunath
  25. University College London, London, UK.

    • Laurence Lovat
    • , Rehan Haidry
    • , Haroon Miah
    • , Sarah Kerr
    • , Victor Eneh
    •  & Rommel Butawan
  26. Norfolk and Waveney Cellular Pathology Network, Norwich, UK.

    • Laszlo Igali
  27. Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

    • Hugo Ford
    • , David Gilligan
    • , Peter Safranek
    • , Andy Hindmarsh
    • , Vijayendran Sudjendran
    • , Andy Metz
    •  & Nick Carroll
  28. Department of Pathology, Wythenshawe Hospital, Manchester, UK.

    • Michael Scott
  29. Edinburgh Cancer Research Centre, Edinburgh University, Edinburgh, UK.

    • J Robert O'Neil

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Contributions

R.C.F. obtained funding and conceived and supervised the study. M.D.E. undertook and supervised the development of the whole-genome analysis pipeline. M.J.D., N.S., A.G.L., M.L.S., B.C. and C.L.A. undertook development of the whole-genome analysis pipeline. S.T., P.A.W.E., N.R., M.D.E., J.M.J.W., C.S.R.-I., N.S. and A.G.L. designed various aspects of the study. J.M.J.W., C.S.R.-I., T.F., M.B. and P.L.-S. extracted the samples and performed the molecular analyses. M.O. performed histopathological diagnosis. T.J.U., N.G., R.H., C.-A.J.O. and L.S. identified and collected samples. J.M.J.W., C.S.R.-I., N.S., A.G.L., M.M., M.J.D., M.L.S., C.L.A., B.C. and M.D.E. analyzed the data. J.M.J.W. performed the analysis of mutational context. A.P.M. designed the Fluidigm primers. J.D. developed the clinical database. C.C., A.O. and S.A. developed the strategy for and performed the verification experiments. J.M.J.W., C.S.R.-I., N.S., A.G.L. and R.C.F. wrote the manuscript. All authors approved the final version of the manuscript.

Competing interests

R.C.F. developed the Cytosponge technology with MRC-Technology, through whom devices were provided for research. The technology has recently been licensed to Covidien; R.C.F. has no direct financial relationship with Covidien.

Corresponding author

Correspondence to Rebecca C Fitzgerald.

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https://doi.org/10.1038/ng.3013

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