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Whole-genome sequencing provides new insights into the clonal architecture of Barrett's esophagus and esophageal adenocarcinoma

Nature Genetics volume 47, pages 1038–1046 (2015)Cite this article

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Abstract

The molecular genetic relationship between esophageal adenocarcinoma (EAC) and its precursor lesion, Barrett's esophagus, is poorly understood. Using whole-genome sequencing on 23 paired Barrett's esophagus and EAC samples, together with one in-depth Barrett's esophagus case study sampled over time and space, we have provided the following new insights: (i) Barrett's esophagus is polyclonal and highly mutated even in the absence of dysplasia; (ii) when cancer develops, copy number increases and heterogeneity persists such that the spectrum of mutations often shows surprisingly little overlap between EAC and adjacent Barrett's esophagus; and (iii) despite differences in specific coding mutations, the mutational context suggests a common causative insult underlying these two conditions. From a clinical perspective, the histopathological assessment of dysplasia appears to be a poor reflection of the molecular disarray within the Barrett's epithelium, and a molecular Cytosponge technique overcomes sampling bias and has the capacity to reflect the entire clonal architecture.

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Figure 1: Paired Barrett's and EAC samples have varied overlap.
Figure 2: EAC samples display multiple copy number changes in comparison to paired Barrett's esophagus samples.
Figure 3: The mutational context is similar for early and late SNVs.
Figure 4: Summary of the samples sequenced for patient AHM1051.
Figure 5: Six distinct clones are present in the non-dysplastic Barrett's esophagus of patient AHM1051.
Figure 6: Multiple different clones can give rise to dysplasia.

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Acknowledgements

We thank the Human Research Tissue Bank, which is supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. This study was partly funded by a project grant from Cancer Research UK. R.C.F. has programmatic funding from the Medical Research Council and infrastructure support from the Biomedical Research Centre and the Experimental Medicine Centre. We would like to thank all the patients who took part in the study. We thank M. Dunning for his bioinformatics assistance. We thank the Edinburgh Experimental Cancer Medicine Centre.

Author information

Author notes

  1. Caryn S Ross-Innes, Jennifer Becq and Suzy Lishman: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Cancer Unit, Hutchison/Medical Research Council Research Centre, University of Cambridge, Cambridge, UK

    Caryn S Ross-Innes, Massimiliano di Pietro, Jamie M J Weaver, Rebecca C Fitzgerald, Barbara Nutzinger, Zarah Abdullahi, Jason Crawte, Shona MacRae, Ayesha Noorani, Rachael Fels Elliott, Jan Bornschein, Tsun-Po Yang, Pierre Lao-Sirieix, Nicola Grehan & Laura Smith

  2. Illumina, Chesterford Research Park, Little Chesterford, UK

    Jennifer Becq, Andrew Warren, R Keira Cheetham, Helen Northen, Sergii Ivakhno, Miao He, Zoya Kingsbury, Mark Ross, Sean Humphray & David Bentley

  3. Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK

    Maria O'Donovan, Shalini Malhotra, Alison Cluroe, Ahmad Miremadi & Betania Mahler-Araujo

  4. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    Andy G Lynch, Lawrence Bower, Paul Edwards, Simon Tavare, Matthew Eldridge & Maria Secrier

  5. Salford Royal NHS Foundation Trust, Salford, UK

    Stephen J Hayes & Yeng Ang

  6. Faculty of Medical and Human Sciences, University of Manchester, UK

    Stephen J Hayes & Yeng Ang

  7. University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK

    Ian Welch

  8. Royal Surrey County Hospital NHS Foundation Trust, Guildford, UK

    Shaun Preston & Sarah Oakes

  9. Edinburgh Royal Infirmary, Edinburgh, UK

    Vicki Save & Richard Skipworth

  10. University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

    Olga Tucker

  11. Department of Computer Science, University of Oxford, UK

    Jim Davies, Charles Crichton & Christian Schusterreiter

  12. Southampton General Hospital, University Hospital Southampton NHS Foundation Trust, Southampton, UK

    Tim Underwood, Fergus Noble, Bernard Stacey, Jamie Kelly, James Byrne, Annette Haydon, Donna Sharland & Jack Owsley

  13. Gloucestershire Royal Hospital, Gloucestershire Hospitals NHS Foundation Trust, Gloucester, UK

    Hugh Barr

  14. Guy's and St Thomas' NHS Foundation Trust, London, UK

    Jesper Lagergren, James Gossage, Andrew Davies, Robert Mason, Fuju Chang, Janine Zylstra & Chris Peters

  15. King's College Hospital, King's College Hospital NHS Foundation Trust, London, UK

    Jesper Lagergren, James Gossage, Andrew Davies, Robert Mason, Fuju Chang & Janine Zylstra

  16. Karolinska Institutet, Stockholm, Sweden

    Jesper Lagergren

  17. Plymouth Hospitals NHS Trust, Plymouth, UK

    Grant Sanders, Tim Wheatley, Richard Berrisford, Tim Bracey, Catherine Harden & David Bunting

  18. Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK

    Tom Roques, Jenny Nobes, Suat Loo, Mike Lewis, Ed Cheong & Oliver Priest

  19. 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

  20. University College London Hospitals NHS Foundation Trust, London, UK

    Laurence Lovat, Rehan Haidry, Haroon Miah, Sarah Kerr, Victor Eneh & Rommel Butawan

  21. Norfolk and Norwich University Hospital, NHS Foundation Trust, Norwich, UK

    Tom Roques, Michael Lewis, Edward Cheong, Bhasker Kumar, Laszlo Igali, Sharon Walton & Adela Dann

  22. Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK

    Peter Safranek, Andy Hindmarsh & Vijayendran Sudjendran

  23. Department of Pathology, Wythenshawe Hospital, Manchester, UK

    Michael Scott

  24. Edinburgh University, Edinburgh, UK

    J. Robert O'Neill & Kasia Adamczuk

  25. Peterborough Hospitals NHS Trust, Peterborough, UK

    Suzy Lishman

  26. University Hospital of North Midlands, NHS Foundation Trust, Stafford, UK

    Duncan Beardsmore & Sarah Dawson

Authors
  1. Caryn S Ross-Innes
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  2. Jennifer Becq
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  3. Andrew Warren
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  5. Helen Northen
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  6. Maria O'Donovan
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  7. Shalini Malhotra
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  8. Massimiliano di Pietro
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  9. Sergii Ivakhno
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  10. Miao He
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  11. Jamie M J Weaver
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  17. Rebecca C Fitzgerald
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Consortia

for the Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) Study Group

  • Stephen J Hayes
  • , Yeng Ang
  • , Ian Welch
  • , Shaun Preston
  • , Sarah Oakes
  • , Vicki Save
  • , Richard Skipworth
  • , Olga Tucker
  • , Jim Davies
  • , Charles Crichton
  • , Christian Schusterreiter
  • , Tim Underwood
  • , Fergus Noble
  • , Bernard Stacey
  • , Jamie Kelly
  • , James Byrne
  • , Annette Haydon
  • , Donna Sharland
  • , Jack Owsley
  • , Hugh Barr
  • , Jesper Lagergren
  • , James Gossage
  • , Andrew Davies
  • , Robert Mason
  • , Fuju Chang
  • , Janine Zylstra
  • , Grant Sanders
  • , Tim Wheatley
  • , Richard Berrisford
  • , Tim Bracey
  • , Catherine Harden
  • , David Bunting
  • , Tom Roques
  • , Jenny Nobes
  • , Suat Loo
  • , Mike Lewis
  • , Ed Cheong
  • , Oliver Priest
  • , Simon L Parsons
  • , Irshad Soomro
  • , Philip Kaye
  • , John Saunders
  • , Vincent Pang
  • , Neil T Welch
  • , James A Catton
  • , John P Duffy
  • , Krish Ragunath
  • , Laurence Lovat
  • , Rehan Haidry
  • , Haroon Miah
  • , Sarah Kerr
  • , Victor Eneh
  • , Rommel Butawan
  • , Tom Roques
  • , Michael Lewis
  • , Edward Cheong
  • , Bhasker Kumar
  • , Laszlo Igali
  • , Sharon Walton
  • , Adela Dann
  • , Peter Safranek
  • , Andy Hindmarsh
  • , Vijayendran Sudjendran
  • , Michael Scott
  • , Alison Cluroe
  • , Ahmad Miremadi
  • , Betania Mahler-Araujo
  • , Barbara Nutzinger
  • , Chris Peters
  • , Zarah Abdullahi
  • , Jason Crawte
  • , Shona MacRae
  • , Ayesha Noorani
  • , Rachael Fels Elliott
  • , Lawrence Bower
  • , Paul Edwards
  • , Simon Tavare
  • , Matthew Eldridge
  • , Jan Bornschein
  • , Maria Secrier
  • , Tsun-Po Yang
  • , J. Robert O'Neill
  • , Kasia Adamczuk
  • , Pierre Lao-Sirieix
  • , Nicola Grehan
  • , Laura Smith
  • , Suzy Lishman
  • , Duncan Beardsmore
  •  & Sarah Dawson

Contributions

R.C.F. conceived the overall study and takes responsibility for the data integrity. C.S.R.-I., J.B. and R.K.C. analyzed the data. C.S.R.-I. extracted the samples for patient AHM1051. A.W. developed the targeted sequencing data visualization tool. C.S.R.-I., J.B., R.K.C., H.N., J.M.J.W., M.R., S.H., D.B. and R.C.F. designed various aspects of the study. H.N. performed the TruSeq Custom Amplicon (TSCA) assay. C.S.R.-I., J.B. and A.G.L. performed the statistical analysis. M.d.P. collected endoscopic samples for patient AHM1051. M.O'D. and S.M. performed the histopathological diagnosis. S.I. developed the copy number pipeline, and R.K.C. and M.H. performed the copy number analysis. Z.K. ran the whole-genome sequencing of patient AHM1051. R.C.F., S.H., D.B. and M.R. supervised the study. C.S.R.-I., J.B., R.K.C. and R.C.F. wrote the manuscript. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Rebecca C Fitzgerald.

Ethics declarations

Competing interests

R.C.F. developed the Cytosponge technology, which has been licensed by MRC-Technology to Covidien. R.C.F. has no direct pecuniary interest. J.B., A.W., R.K.C., H.N., S.I., M.H., Z.K., M.R., S.H. and D.B. are employees of Illumina.

Integrated supplementary information

Supplementary Figure 1 Multiple samples from five patients (P4, P8, P14, P15 and P17) with Barrett's esophagus and adjacent EAC show that the poor overlap is not a result of sampling bias.

Bar graphs showing the number of SNVs for samples of Barrett's esophagus (BE) and cancer (C) that are either present in all samples (teal), present in all BE samples and absent in all cancer samples (pink), or present in all cancer samples and absent in all BE samples (gray) for each particular patient. Purple bars show the number of remaining variants not accounted for in the other groups (for example, present in a proportion of samples).

Supplementary Figure 2 EAC samples display multiple copy number aberrations compared to paired Barrett's samples.

(a) Copy number plots showing two examples of Barrett's esophagus genomes that are majority copy number 2, compared to the matched EAC samples, which contain multiple gains and losses. (b) Similar plots but for patients where copy number events can be seen in the Barrett's esophagus sample as well as in the EAC sample (but are not significant enough to make a call). (c) Examples of copy number events in the Barrett's esophagus sample that are not seen in the matching EAC sample. The top track shows the depth ratio in a 20-kb window, and the bottom track shows the B-allele ratio.

Supplementary Figure 3 Principal-component analysis of mutational context.

The counts of each different mutational context in each of the three sets of SNVs (common, Barrett's esophagus (BE) unique, EAC unique) were used as input for a principal-component analysis. There is some separation between the common SNVs and the unique SNVs in the second component, but the differences between the groups are not striking (6.9% variance in the second component). (b) Plot showing the weights of the 96 different mutational contexts. Four mutational contexts—C(A>C)C, G(C>G)G and C(A>T)C (all at the top right-hand side of the plot) as well as T(C>G)A (bottom right)—seem to be the main cause for the separation in the second component.

Supplementary Figure 4 Copy number plot for one of the Barrett's esophagus samples with low-grade dysplasia.

Copy number plot for one of the Barrett's esophagus samples with low-grade dysplasia that was sent for whole-genome sequencing. Large deletions on multiple chromosomes, including chromosomes 5, 11, 13, 18 and 21, can be seen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–4. (PDF 1340 kb)

Supplementary Data Set

Nebula: custom-made data browser to investigate and visualize the results from the 1,443 targets that were sequenced for 73 Barrett's esophagus samples from patient AHM1051. (ZIP 7637 kb)

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Ross-Innes, C., Becq, J., Warren, A. et al. Whole-genome sequencing provides new insights into the clonal architecture of Barrett's esophagus and esophageal adenocarcinoma. Nat Genet 47, 1038–1046 (2015). https://doi.org/10.1038/ng.3357

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