Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis

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.

Figure 1. Flow chart illustrating the study outline

The number of samples used at each stage is given. The methodology used for each study phase is shown on the left hand side. EAC, Esophageal adenocarcinoma, BE, Barrett’s esophagus, HGD, high grade dysplasia.

Figure 2. Mutation in esophageal adenocarcinoma

The bar graph on the top indicates the percentage of samples with aberrations for a given gene. The number in bold denotes the total number of mutations for each gene. Genes with four or more mutations in our EAC discovery and validation cohort (combined total of 112 patients) were included. The proportion of missense, nonsense/splice and indel mutations are shown. The matrix below shows the number of samples with mutations in both genes for each possible pairing of genes. The red highlighted box indicates significantly co-occurring mutations (Significance was assessed empirically from 100,000 permutations. False discovery rate was controlled using the Benjamini-Hochberg procedure.).

Figure 3. TP53 and SMAD4 mutations accurately define the boundaries in the progression towards cancer whilst other mutations appear to occur independent of disease stage

A. Bar graph showing the number of never-dysplastic Barrett’s esophagus patients (NDBE) (n=40), Barrett’s esophagus patients with high grade dysplasia (HGD) (n=39) and EAC patients (n=112) with at least one mutation in our panel consisting of 26 genes. B. Percentage of never-dysplastic Barrett’s esophagus, Barrett’s esophagus with HGD and EAC samples with mutations in recurrently-mutated genes (mutated in ≥4 samples) identified in the EAC discovery cohort and EAC Validation cohort. TP53 and SMAD4 are the only genes for which mutations separate the boundaries between never-dysplastic and dysplastic Barrett’s esophagus (TP53) or cancer (SMAD4) (two-tailed Fisher’s exact test with Benjamini-Hochberg correction for multiple testing, * p<0.05). C. Proposed model for the boundary-defining mutations in Barrett’s esophagus carcinogenesis. The hashed box depicts multiple other mutations which may occur and provide selective advantage at any stage of disease.

Figure 4. TP53 mutations can be used to diagnose Barrett’s esophagus with prevalent high-grade dysplasia on the Cytosponge™

A. The allele fraction of TP53 mutations identified in Cytosponge™ samples is shown for the three patients groups: no Barrett’s esophagus (n=23), Barrett’s esophagus with no dysplasia (n=44) and Barrett’s esophagus with high grade dysplasia (HGD) (n=22). B. The positions of the TP53 mutations identified on the Cytosponge™ samples are shown above the gene diagram compared with those found in the EAC and Barrett’s esophagus HGD biopsy cohorts. The dotted line on the gene outline denotes the two small areas not covered by the multiplex PCR assay (amino acids 1-27 and 361-393). TA, transcription activation domain; OD, oligomerization domain.