Targeted genetic and epigenetic profiling of esophageal adenocarcinomas and non-dysplastic Barrett's esophagus

Abstract

Background: Despite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett's esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses.

Results: Overall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21-89%), as well as in a subset of adjacent normal samples (up to 12%).

Conclusions: Our study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.

Keywords: Barrett’s esophagus; DNA methylation; Esophageal adenocarcinoma; Microsatellite instability; TP53 mutations.

Fig. 1

Summary of genetic and epigenetic alterations in BE (n = 19) and EAC (n = 145) samples. TP53 silent mutations with no amino acid change are not presented as alterations. In one sample (patient 83), two TP53 missense mutations were found. All samples with no or a low percentage of tumor cells (< 5%; n = 37) have been removed from the main data set (see Fig. 2), and the molecular alterations found in these samples are shown separately in the gray box. These samples were not used for determination of alterations frequencies. (*For MSI, the percentage refers to MSI-H tumors only.)

Fig. 2

Flow diagram illustrating EAC samples selection process. One hundred and forty-five EAC patients were subjected to targeted molecular profiling, among which 37 were removed from the main data set due to the absence of tumor or low tumor cell content (< 5%). Only samples from 108 patients were used for determination of alterations frequencies

Fig. 3

Graphical representation of TP53 mutations identified in BE and EAC samples. The entire TP53 coding region (exons 2–11) was analyzed by Sanger sequencing, and mutations were found across exons 4–8. The silent mutation R213R found in one BE (patient 13) and one EAC (patient 94) sample was classified as TP53 wild type

Fig. 4

PMR values distribution in BE (n = 19), EAC (n = 108) and respective normal adjacent mucosa (N). The thresholds for scoring the samples as methylated were set according to the highest PMR value across the normal mucosa matching BE samples. These thresholds were determined for each gene independently and are marked by dotted red lines

Fig. 5

Methylation levels of the evaluated genes in BE, EAC (T) and normal adjacent mucosa (N). EAC patients where normal mucosa presents PMR values higher than in the tumor are highlighted by gray bordered boxes. PMR values are shown in different color scales for each gene in order to facilitate visualization