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


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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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

Authors and Affiliations




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.

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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).

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