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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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.
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.
Integrated supplementary information
The bar graph represents the total number of each possible mutation class change. The heat map displays the enrichment of each mutation class at any given trinucleotide context. For example, the strong red strip running down the center of the figure represents enrichment for C:G>T:A mutations at the XpCpG trinucleotide. Enrichment at the CTT trinucleotide can be seen for all three classes of thymidine mutation in the majority of samples.
(a) Schematic showing ARID1A mutations identified in the discovery and validation cohort (112 EAC cases). (b) Examples of two EAC tumors, one staining positively for ARID1A protein expression and one staining negatively for ARID1A protein expression.
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Weaver, J., Ross-Innes, C., Shannon, N. et al. Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis. Nat Genet 46, 837–843 (2014) doi:10.1038/ng.3013
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